Dairy-Derived Ingredients: Food and Nutraceutical Uses (Woodhead Publishing in Food Science, Technology and Nutrition)

Dairy-derived ingredients Food and nutraceutical uses Edited by Milena Corredig

WPKN041109

Oxford

Cambridge

New Delhi

Published by Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, UK www.woodheadpublishing.com Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj New Delhi – 110002, India www.woodheadpublishingindia.com Published in North America by CRC Press LLC, 6000 Broken Sound Parkway, NW, Suite 300, Boca Raton, FL 33487, USA First published 2009, Woodhead Publishing Limited and CRC Press LLC © 2009, Woodhead Publishing Limited except Chapter 6 and Chapter 23 © United States Government; Chapter 5 figures © R. Durham The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress. Woodhead Publishing ISBN 978-1-84569-465-4 (book) Woodhead Publishing ISBN 978-1-84569-719-8 (e-book) CRC Press ISBN 978-1-4398-2070-4 CRC Press order number: N10128 The publishers’ policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elemental chlorine-free practices. Furthermore, the publishers ensure that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by Toppan Best-set Premedia Limited Printed by TJ International Ltd, Padstow, Cornwall, UK

Preface

This book is a great collection of chapters, written by acknowledged experts, providing up-to-date views on dairy ingredients manufacture, interactions and uses. Today milk can no longer be considered as a commodity, but rather as a source of value-added ingredients, both from the processing and the nutritional point of view. In the past few years there has been a fundamental change in the way we process and develop dairy products and ingredients. Our knowledge of the structure and the structure–function changes of dairy components with processing has significantly improved over the past decade, and this has resulted in major advances in the production of ingredients. Membrane filtration, for example, has resulted in significant changes in the availability of whey, casein and milk protein concentrates and isolates, and the optimization of this unit operation has created a number of novel ingredients tailored to specific processing functionalities. Other processes, as for example, use of high pressure, have been studied in great detail, with possible commercial applications perhaps limited only by lack of initial capital investment. Because of the major progress made in chromatographic techniques and membrane separation, isolation of minor components in milk has become a reality, and studies on the functionality of these ingredients are no longer only an academic exercise but an industrial reality. For example, it is possible now to purify immunoglobulins from bovine colostrums and whey on a large scale, to produce concentrates as dietary supplements, and, with the application of high pressure processing to these products, it will be possible to minimize the denaturation of these molecules and therefore optimize their biological functionality.

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Preface

The processing functionality of dairy ingredients continues to remain important, and therefore research efforts continue to be carried out to understand interactions and predict their behaviour during processing. In particular, there is an emerging trend to use dairy systems as platforms for the delivery of bioactive molecules in foods. Many milk components can function as encapsulating agents. Micro and nano-encapsulation can protect active components from the surrounding environment and tailor and control their release. Although the study of the extraction and potential utilization of the various components in milk continues to be an important aspect of dairy research, the focus has shifted in the past decade from processing to nutritional functionality. A new generation of high-value, functional ingredients, not only for the food but also for the pharmaceutical market are being developed, as more and more evidence is brought forward of the beneficial effect of their consumption to human health. The positive health-promoting aspects of dairy ingredients, whey, caseins, milk fat globule membrane material, oligosaccharides and glycoconjugates have been so far tested mostly in vitro or in simple animal models, and more work needs to be carried out to demonstrate their properties. However, the evidence continues to accumulate on antimicrobial, anti-inflammatory, hypocholesterolemic, hypertension controlling, anti-carcinogenic activities of milk-derived products. This book attempts to summarize our current knowledge in some of the most promising areas. In the future, milk ingredients may play a complementary role to the pharmacological drugs in the prevention and treatment of various chronic diseases. Epidemiological studies have linked the consumption of dairy products to alleviation of physiological characteristics associated with diabetes, cardiovascular diseases, metabolic syndrome and obesity; however, the results are often inconsistent, possibly because of the variety of composition of the dairy products consumed. Milk proteins are also considered one of the most important sources of bioactive peptides; however, their isolation and characterization still need to be developed, especially to evaluate the synergy occurring when consuming dairy products. In addition to simple enzymatic hydrolysis, an emerging field is biogenics, the study of the formation of bioactives as a result of tailored fermentations in dairy products. Another area that shows great promise is the study of the biological role of glycoproteins in milk and their potential for applications. Glycans from milk proteins may serve as recognition sites interacting with cell membranes, viruses and microorganisms. There is an increasing evidence that these molecules have beneficial effects within the gastrointestinal tracts, modulate the intestinal flora preventing attachment of pathogens and viruses, and interact directly with immune cells. These molecules could soon find therapeutic applications. Finally, the study of minor milk lipids derived from the milk fat globule membrane is also at its infancy.

Preface

xxi

As will become evident by reading the various chapters of this book, the future of the use of dairy ingredients is an interesting one and we are certainly at a turning point in the development of value-added components from milk. I am very grateful to the all the authors for their hard work on this project and their excellent contributions. Milena Corredig

Contributor contact details

(* = main contact)

Chapter 1 Professor U. Kulozik Food Process Engineering and Dairy Technology Technische Universität München Arcisstrasse 21 80333 München Germany

Chapter 3 L. Vercruysse, Professor J. Van Camp*, Professor K. Dewettinck Department of Food Safety and Food Quality Department of Crop Protection Ghent University Coupure Links 653 9000 Ghent Belgium E-mail: [email protected]

E-mail: [email protected]

Chapter 2 Dr Pierre Schuck INRA Agrocampus Ouest UMR STLO 65, rue de Saint Brieuc 35042 Rennes cedex France E-mail: [email protected]. fr

Professor G. Smagghe and L. Vercruysse Department of Crop Protection Ghent University Coupure Links 653 9000 Ghent Belgium

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Contributor contact details

Chapter 4

Chapter 6

M. Sichien, N. Thienpont, E. Fredrick, T. Trung Le, Professor J. Van Camp and Professor K. Dewettinck* Department of Food Safety and Food Quality (LA07) Faculty of Bioscience Engineering Ghent University Coupure Links 653 9000 Ghent Belgium

Dr P. X. Qi United States Department of Agriculture Agricultural Research Service Eastern Regional Research Center Dairy Processing and Products Research Unit 600 East Mermaid Lane Wyndmoor, PA 19038 USA E-mail: [email protected]

E-mail: Koen.Dewettinck@UGent. be

Chapter 7 N. Thienpont Department of Food Science and Technology Faculty of Biosciences and Landscape Architecture University College Ghent Voskenslaan 270, 9000 Gent, Belgium

Dr I. Recio*, Dr F. J. Moreno and Professor R. López-Fandiño Instituto de Fermentaciones Industriales (CSIC) C/Juan de la Cierva, 3 28006 Madrid Spain E-mail: recio@ifi.csic.es

Chapter 5 Dr R. J. Durham School of Natural Sciences University of Western Sydney Hawkesbury Campus Locked Bag 1797 Sth Penrith, NSW 1797 Australia E-mail: [email protected]

Chapter 8 T. Akhavan, S. Panahi, Professor G. H. Anderson and Dr B. L. Luhovyy* Department of Nutritional Sciences Faculty of Medicine University of Toronto 150 College St., 329a FitzGerald Bldg Toronto, ON Canada M5S 3E2 E-mail: bohdan.luhovyy@utoronto. ca

Contributor contact details

xv

Chapter 9

Chapter 12

Dr Riina Kekkonen* Valio Ltd R&D PO Box 30 FIN-00039 Helsinki Finland

Dr R. Hickey Moorepark Food Research Centre Teagasc Moorepark Fermoy, Co. Cork Ireland E-mail: [email protected]

E-mail: riina.kekkonen@valio.fi Dr Katri Peuhkuri Netnut Espoo Finland Dr Riina Kekkonen and Dr Katri Peuhkuri University of Helsinki Institute of Biomedicine Finland

Chapter 10 Professor H. J. Korhonen* and Dr P. Marnila Biotechnology and Food Research MTT Agrifood Research Finland FIN-31600, Jokioinen Finland E-mail: hannu.j.korhonen@mtt.fi

Chapter 13 Dr S. Mezouari* and Y. Pouliot Department of Food Science and Nutrition Laval University Pavillon Paul-Comtois Quebec City Quebec Canada G1K 7P4 E-mail: [email protected] Dr M. Britten Food Research and Development Centre Agriculture and Agri-Food Canada 3600 Casavant Boulevard West St-Hyacinthe QC Canada J2S 8E3

Chapter 11 Dr P. Marnila* and Professor H. J. Korhonen Biotechnology and Food Research MTT Agrifood Research Finland FIN-31600, Jokioinen Finland E-mail: pertti.marnila@mtt.fi

Chapter 14 Dr M. Corredig Department of Food Science University of Guelph Guelph, Ontario Canada N1H 2W5 E-mail: [email protected]

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Contributor contact details

Chapter 15

Chapter 18

Dr T. Huppertz NIZO food research P.O. Box 20 6710BA Ede The Netherlands

Dr M. Sibeijn Process technology engineer Dishman Netherlands B.V. Veenendaal The Netherlands

E-mail: [email protected]

Dr J.A. Wouters* NIZO Food Research P.O. Box 20 6710 BA Ede The Netherlands

Chapter 16 Professor A. L. Kelly*, Dr K. I. Kothari, Dr D. D. Voigt Department of Food and Nutritional Sciences University College Cork Ireland E-mail: [email protected] Dr T. Huppertz NIZO Food Research Ede The Netherlands

Chapter 17 Professor M. A. Drake*, J. Wright, M. Whitson and M. Lloyd Dept. Food, Bioprocessing, and Nutritional Sciences Southeast Dairy Foods Research Center North Carolina State University Raleigh, NC 27695 USA E-mail: [email protected]

E-mail: [email protected]

Chapter 19 Dr B. T. O’Kennedy Moorepark Food Research Centre Teagasc Moorepark Fermoy Co. Cork Ireland E-mail: Brendan.okennedy@ teagasc.ie

Chapter 20 T. P. Guinee Moorepark Food Research Centre Teagasc Moorepark Fermoy Co. Cork Ireland E-mail: [email protected]

Contributor contact details

Chapter 21

Chapter 24

Dr P. J. Wilde Institute of Food Research Norwich NR4 7UA UK

Dr A. H. J. Paterson* School of Engineering and Advanced Technology Massey University Private Bag 11 222 Palmerston North New Zealand

E-mail: [email protected]

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E-mail: [email protected]

Chapter 22 Dr C. M. Oliver and Dr M.-A. Augustin* CSIRO Preventative Health Flagship CSIRO Division of Food and Nutritional Sciences 671 Sneydes Road Werribee Victoria 3030 Australia E-mail: [email protected]

Chapter 23 Dr P. M. Tomasula United States Department of Agriculture Agricultural Research Service Eastern Regional Research Center Dairy Processing and Products Research Unit 600 E. Mermaid Lane Wyndmoor, PA 19038 USA E-mail: [email protected]. gov

Dr S. J. Kellam Hilmar Cheese Company 9001 N. Lander Avenue PO BOX 910 Hilmar, CA 95324 USA E-mail: skellam@hilmaringredients. com

Chapter 25 Professor Harjinder Singh Riddet Institute Massey University Private Bag 11222 Palmerston North New Zealand E-mail: [email protected]

Contents

Contributor contact details......................................................................... Preface..........................................................................................................

Part I

1

2

Modern approaches to the separation of dairy components and manufacture of dairy ingredients .................

Novel approaches for the separation of dairy components and manufacture of dairy ingredients ..................................................... U. Kulozik, Technische Universität München, Germany 1.1 Introduction............................................................................ 1.2 State-of-the-art separation techniques ............................... 1.3 Process intensification by hybrid processes ....................... 1.4 Alternative membrane processing concepts ...................... 1.5 References .............................................................................. Understanding the factors affecting spray-dried dairy powder properties and behavior..................................................................... P. Schuck, INRA, France 2.1 Introduction............................................................................ 2.2 Principles of spray drying ..................................................... 2.3 Properties and qualities of dairy powders ......................... 2.4 Effects of spray drying parameters on powder properties ................................................................................ 2.5 Control and improvement of powder properties .............. 2.6 Conclusions and future trends ............................................. 2.7 References ..............................................................................

xiii xix

1

3 3 6 11 17 23

24 24 26 32 39 44 47 48

vi 3

4

5

Contents Production and enrichment of bioactive peptides derived from milk proteins .............................................................................. L. Vercruysse, J. Van Camp, K. Dewettinck and G. Smagghe, Ghent University, Belgium 3.1 Introduction............................................................................ 3.2 Enzymatic hydrolysis ............................................................ 3.3 Microbial fermentation ......................................................... 3.4 Enrichment of bioactive peptides ....................................... 3.5 Future trends .......................................................................... 3.6 Acknowledgement................................................................. 3.7 Sources of further information and advice ........................ 3.8 References .............................................................................. Processing means for milk fat fractionation and production of functional compounds ................................................................... M. Sichien, N. Thienpont, E. Fredrick, T. Trung Le, J. Van Camp and K. Dewettinck, Ghent University, Belgium 4.1 Introduction............................................................................ 4.2 Overview of milk lipids ........................................................ 4.3 Milk fat modification ............................................................ 4.4 Milk fat minor components ................................................. 4.5 Nutritional properties of dairy fats ..................................... 4.6 Acknowledgement................................................................. 4.7 References .............................................................................. Modern approaches to lactose production ..................................... R. J. Durham, University of Western Sydney, Australia 5.1 Introduction............................................................................ 5.2 Whey and permeate pretreatments .................................... 5.3 Lactose crystallisation processes ......................................... 5.4 Post handling – separation, washing and drying crystals..................................................................................... 5.5 Specialised pharmaceutical products .................................. 5.6 Lactose standards and specifications .................................. 5.7 Mother liquor ......................................................................... 5.8 Applications and functionality of lactose .......................... 5.9 Current and future trends .................................................... 5.10 Sources of further information and advice ........................ 5.11 References ..............................................................................

Part II

6

51

51 53 55 58 62 62 63 63

68

68 69 72 86 94 95 95 103 103 108 119 125 127 129 130 132 133 134 135

Biological functionality of dairy components and nutraceutical applications ..........................................................

145

Studies of the biological function and structure of casein micelles, and future implications ......................................................

147

Contents P. X. Qi, Agricultural Research Service, USDA, USA 6.1 Introduction............................................................................ 6.2 Brief review of proposed models for casein micelles ....... 6.3 Synthesis and secretion of caseins....................................... 6.4 Studies on the structure of casein micelles ........................ 6.5 Future trends .......................................................................... 6.6 Acknowledgements ............................................................... 6.7 References ..............................................................................

vii

147 148 152 160 163 164 164

7

Glycosylated dairy components: Their roles in nature and ways to make use of their biofunctionality in dairy products .................... 170 I. Recio, F. J. Moreno and R. López-Fandiño, Instituto de Fermentaciones Industriales (CSIC), Spain 7.1 Introduction: Glycosylation of milk proteins .................... 170 7.2 κ-casein and caseinmacropeptide ........................................ 171 7.3 Glycosylated whey proteins ................................................. 178 7.4 Glycoproteins associated with the milk-fat globule membrane (MFGM) ............................................................. 185 7.5 Conclusions and future trends ............................................. 196 7.6 Acknowledgements ............................................................... 196 7.7 References .............................................................................. 196

8

Application of dairy-derived ingredients in food intake and metabolic regulation .......................................................................... T. Akhavan, S. Panahi, G. H. Anderson and B. L. Luhovyy, University of Toronto, Canada 8.1 Introduction............................................................................ 8.2 Dairy-derived ingredients..................................................... 8.3 Relationship between milk and chronic diseases.............. 8.4 Dairy components and their effect on satiety and food intake regulation .......................................................... 8.5 Dairy-based functional foods. .............................................. 8.6 Conclusion .............................................................................. 8.7 Notation .................................................................................. 8.8 References ..............................................................................

9

Bioactive milk protein and peptide functionality .......................... R. Kekkonen, Valio Ltd, Finland and University of Helsinki, Institute of Biomedicine, Finland; and K. Peuhkuri, Netnut, Finland and University of Helsinki, Institute of Biomedicine, Finland 9.1 Introduction............................................................................ 9.2 Major bioactive milk proteins and peptides ...................... 9.3 Health-promoting properties of bioactive milk proteins and peptides ............................................................ 9.4 Future trends ..........................................................................

212

212 213 220 224 227 229 229 229 238

238 239 242 254

viii

Contents 9.5 9.6

10

11

12

13

Sources of further information and advice ........................ References ..............................................................................

Bovine milk immunoglobulins against microbial human diseases................................................................................................. H. J. Korhonen and P. Marnila, MTT Agrifood Research Finland, Finland 10.1 Introduction............................................................................ 10.2 Properties of immunoglobulins ........................................... 10.3 Production and isolation of immunoglobulins .................. 10.4 Applications of immunoglobulins ....................................... 10.5 Future trends .......................................................................... 10.6 References .............................................................................. Lactoferrin for human health ........................................................... P. Marnila and H. J. Korhonen, MTT Agrifood Research Finland, Finland 11.1 Introduction............................................................................ 11.2 Antimicrobial activities......................................................... 11.3 Cancer prevention and immunological effects .................. 11.4 Other biological activities .................................................... 11.5 Applications and safety aspects........................................... 11.6 References .............................................................................. Harnessing milk oligosaccharides for nutraceutical applications.......................................................................................... R. Hickey, Moorepark Food Research Centre, Teagasc, Ireland 12.1 Introduction............................................................................ 12.2 Composition of oligosaccharides in milk ........................... 12.3 Nutraceutical potential of milk oligosaccharides .............. 12.4 Isolation of milk oligosaccharides for use as food additives .................................................................................. 12.5 Commercial products containing milk oligosaccharides ..................................................................... 12.6 Perspectives and future trends ............................................ 12.7 References .............................................................................. Lipids from the milk fat globule membrane as a health ingredient: Composition, properties and technological aspects .................................................................................................. S. Mezouari and Y. Pouliot, Laval University, Canada; M. Britten, Agriculture and Agri-Food Canada, Canada 13.1 Introduction............................................................................ 13.2 Composition of the milk fat globule membrane lipids .... 13.3 Separation of milk fat globule membrane lipids...............

254 255

269

269 270 275 277 284 285 290

290 291 295 297 298 302

308

308 310 312 329 330 331 331

344

344 345 353

Contents 13.4 13.5 13.6 13.7

Part III

14

15

16

ix

Effect of processing on milk fat globule membrane lipids ........................................................................................ Beneficial effects of milk fat globule membrane lipids ........................................................................................ Conclusion and perspectives ................................................ References ..............................................................................

356 358 358

Technological functionality of dairy components, and food and non-food applications ......................................

369

Molecular understanding of the interaction of dairy proteins with other food biopolymers ............................................. M. Corredig, University of Guelph, Canada 14.1 Introduction............................................................................ 14.2 Interpolymer complexes: Interactions of polysaccharides with whey proteins or sodium caseinate .................................................................... 14.3 Interactions of casein micelles with polysaccharides ...................................................................... 14.4 Polysaccharides interactions with milk proteins adsorbed at oil–water interfaces ......................................... 14.5 Conclusions ............................................................................ 14.6 References .............................................................................. Novel applications of enzymes in the dairy sector: Optimizing functional properties of milk proteins by enzymatic cross-linking......................................................................................... T. Huppertz, NIZO food research, The Netherlands 15.1 Introduction............................................................................ 15.2 Functional properties of milk proteins ............................... 15.3 Enzymatic cross-linking of milk proteins ........................... 15.4 Physicochemical properties of cross-linked milk proteins ................................................................................... 15.5 Gelation properties of milk systems subjected to enzymatic cross-linking ......................................................... 15.6 Emulsification properties of cross-linked milk proteins ................................................................................... 15.7 Conclusions ............................................................................ 15.8 References .............................................................................. Improving technological and functional properties of milk by high-pressure processing .............................................................. A. L. Kelly, K. I. Kothari and D. D. Voigt, University College Cork, Ireland; T. Huppertz, NIZO food research, The Netherlands

354

371 371

374 378 383 388 388

394 394 395 398 403 405 410 411 412

417

x

Contents 16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 16.9

17

18

19

Introduction to high-pressure processing in the food industry ................................................................... Effects of high-pressure treatment on constituents and properties of milk .......................................................... Applications of high-pressure treatment in processing liquid milk ........................................................... High-pressure treatment of milk for the manufacture of acid-coagulated milk products ................. High-pressure treatment of milk for cheese manufacture............................................................................ High-pressure treatment of milk for ice-cream manufacture............................................................................ Commercial prospects for high-pressure processing in the dairy industry .............................................................. Conclusion .............................................................................. References ..............................................................................

Impact of dairy ingredients on the flavor profiles of foods .......... M. A. Drake, J. Wright, M. Whitson and M. Lloyd, North Carolina State University, USA 17.1 Defining flavor of dried ingredients.................................... 17.2 Sources of flavors in dried dairy ingredients ..................... 17.3 Flavor carry-through with dried dairy ingredients ........... 17.4 Future trends .......................................................................... 17.5 References .............................................................................. Production of dairy aromas and flavors: New directions .............. M. Sibeijn, Dishman Netherlands B.V., The Netherlands and J. A. Wouters, NIZO food research, The Netherlands 18.1 Introduction............................................................................ 18.2 Production methods for dairy aromas and flavors ........... 18.3 Downstream processing of dairy aromas and flavors....... 18.4 Future trends .......................................................................... 18.5 References .............................................................................. Dairy ingredients in non-dairy food systems .................................. B. T. O’Kennedy, Moorepark Food Research Centre, Teagasc, Ireland 19.1 Introduction to dairy ingredients and their potential use in other food systems .................................... 19.2 Functionality of dairy ingredients ....................................... 19.3 Dairy ingredients in food systems ....................................... 19.4 Bakery products..................................................................... 19.5 Future trends .......................................................................... 19.6 References ..............................................................................

417 419 425 426 428 432 432 434 435 442

442 443 463 465 466 470

470 471 472 476 480 482

482 489 493 502 503 504

Contents 20

21

22

23

The role of dairy ingredients in processed cheese products ............................................................................................... T. P. Guinee, Moorepark Food Research Centre, Teagasc, Ireland 20.1 Introduction............................................................................ 20.2 Processed cheese types and permitted ingredients........... 20.3 Manufacture of processed cheese products ....................... 20.4 Effects of different ingredients on properties of processed cheese products ................................................... 20.5 Conclusions ............................................................................ 20.6 References .............................................................................. Emulsions and nanoemulsions using dairy ingredients................. P. J. Wilde, Institute of Food Research, UK 21.1 Introduction............................................................................ 21.2 Emulsion formation and stability ........................................ 21.3 Dairy ingredients as emulsifiers .......................................... 21.4 Effects of food processing .................................................... 21.5 Improving functionality ........................................................ 21.6 Future trends .......................................................................... 21.7 Sources of further information and advice ........................ 21.8 References .............................................................................. Using dairy ingredients for encapsulation ...................................... C. M. Oliver and M. A. Augustin, CSIRO Division of Food and Nutritional Sciences, Australia 22.1 Introduction............................................................................ 22.2 Designing encapsulated ingredients.................................... 22.3 Encapsulating properties of dairy ingredients .................. 22.4 Encapsulated systems utilizing dairy ingredients as encapsulants ........................................................................... 22.5 Future trends .......................................................................... 22.6 Sources of further information and advice ........................ 22.7 References .............................................................................. Using dairy ingredients to produce edible films and biodegradable packaging materials.................................................. P. M. Tomasula, Agricultural Research Service, USDA, USA 23.1 Introduction............................................................................ 23.2 The milk proteins used for film formation ........................ 23.3 Edible films and coatings made from casein or whey proteins ......................................................................... 23.4 Improvements to edible films .............................................. 23.5 Milk proteins in composite films .........................................

xi

507

507 509 511 517 531 533 539 539 541 547 553 557 558 559 559 565

565 566 569 570 580 581 582

589

589 590 593 607 613

xii

Contents 23.6 23.7 23.8 23.9 23.10

24

25

Modifying the properties of edible films through processing ............................................................................... Potential applications ............................................................ Future trends .......................................................................... Sources of further information and advice ........................ References ..............................................................................

Transformation of lactose for value-added ingredients ................ A. H. J. Paterson, Massey University, New Zealand; S. J. Kellam, Hilmar Cheese Company, USA 24.1 Introduction............................................................................ 24.2 Galacto-oligosaccharides ...................................................... 24.3 Lactulose................................................................................. 24.4 Lactitol .................................................................................... 24.5 Lactobionic acid..................................................................... 24.6 Hydrolyzed lactose ................................................................ 24.7 Tagatose .................................................................................. 24.8 Lactose as a fermentation substrate ................................... 24.9 References .............................................................................. Protein interactions and functionality of milk protein products ............................................................................................... H. Singh, Massey University, New Zealand 25.1 Introduction............................................................................ 25.2 Milk protein products and powders.................................... 25.3 Protein interactions during the manufacture of skim milk powder and milk protein concentrate powder ......... 25.4 Protein interactions during the manufacture of whey protein and casein products ................................................. 25.5 Functional properties and applications of milk protein products .................................................................................. 25.6 References ..............................................................................

Index .............................................................................................................

614 615 616 618 619 625

625 631 631 632 633 634 635 637 638

644 644 646 650 656 659 669 675

Part I Modern approaches to the separation of dairy components and manufacture of dairy ingredients

1 Novel approaches for the separation of dairy components and manufacture of dairy ingredients U. Kulozik, Technische Universität München, Germany

Abstract: Novel approaches regarding the separation of dairy components are discussed in the light of continuous efforts to better understand boundary layer phenomena in membrane separation. For instance, deposit formation by proteins can be minimized only through crossflow filtration when the fluid dynamics along the membrane surface are under control and chemico-physical effects controlling adsorption are understood. Thus, even existing processes can be intensified by means of more appropriate processing and milieu conditions, together with selecting membranes with optimal membrane resistances relative to individual application. Hybrid processes and novel techniques such as membrane adsorption or electro assisted filtration are described as ways to expand the reach of current membrane technologies. Key words: membrane filtration, boundary layer phenomena, membrane adsorption chromatography, dynamic membrane systems, electric field assisted membrane techniques.

1.1

Introduction

Fractionation of milk components has traditionally been a strong foundation for the production of a huge variety of dairy products. Centrifugation is applied to separate cream and skim milk. Coagulation is induced to separate protein from the aqueous phase in the manufacture of cheeses. Chromatographic techniques have been established on an industrial scale for the removal of lactose from milk to produce low lactose or lactose-free milk in Finland and other countries. This chapter focuses mainly on more recent insights and new developments in research, and on the application of membrane technologies in the field of dairy production, with membranes applied alone or in combination with other processes.

4

Dairy-derived ingredients

Dairy components of relevance for separation and fractionation by means of membrane technologies are major and minor proteins in the first place and low molecular weight components such as ionic substances and lactose. All these components are of influence in membrane separation because of their molecular size or surface properties. Proteins, but also fat globule membrane components, are macromolecules prone to adsorption and formation of gel deposits on membrane surfaces. Ionic substances and lactose are small and therefore can pass through porous membranes. In contrast to that, they cannot pass through non-porous, homogeneous membranes, typical for reverse osmosis, and are therefore mainly responsible for the osmotic pressure of milk and whey. The osmotic pressure is a key intrinsic factor of the product influencing flux in reverse osmosis. The retention or passage of components nominally depends on the membrane pore size and structure. However, it is practically experienced that apart from size, other properties of molecules, such as charge, structure, and shape, play decisive roles in this respect. Beyond molecular weight, for instance, proteins vary in their surface charge and hydrodynamic volume or diameter as a function of pH and ionic composition of the aqueous phase environment. Depending on the physical properties of the membrane and other solutes, this often results in unspecific interactions of compounds with the membrane surface, causing adsorption or deposited layers, and these effects are difficult to predict. An example is given in Fig. 1.1, where the impact of pH on the permeation of β-lactoglobulin through a microfiltration membrane is depicted. As can be seen, the permeation qualitatively follows the changes occurring to the quaternary structure of β-lactoglobulin, which, depending on pH, exists as a monomer, dimer or octamer. The practical implication of this effect in dairy technology is that β-lactoglobulin should have different permeation patterns in the filtration of acid or sweet whey if microfiltration is applied for the reduction of microorganisms in whey prior to the production of WPC products. Another key factor is the ionic composition of the solution, which has an impact on the surface charge of colloidal particles such as proteins, including whey proteins and casein micelles. This is schematically shown in Fig. 1.2, where the effect of low or high ionic strengths on the electric double layer of the particles is described. The electrical double layer around charged particles (proteins carry a net charge depending on the composition of charged amino acids) is thin in high ionic environments, and high at lower ionic strengths. Thus, the hydrodynamic size of molecules is not constant – it rather depends on environmental factors such as pH and ionic composition, i.e. on electrical shielding effects and collapse of the hairy layer of protein residues protruding from the casein micelle into the outer phase. In conclusion, despite its long history of application in dairy technology and strong impact on production methods, membrane separation is still a complex and not fully understood field because of various factors influen-

Novel approaches for the separation of dairy components

5

70

50 40 30

6.9 nm

Permeation of β-Lg (%)

60

20 3.6 nm 10 2

3

4

5

6

7

8

pH

Electrostatic potential around a charged particle

Fig. 1.1 Permeation (p) of β-lactoglobulin (molecular diameter approx. 3–4 nm) through a ceramic microfiltration membrane (nominal pore size 0.1 µm) as a function of pH. p is defined as the ratio of the β-lactoglobulin concentration found in the permeate relative to the concentration in the retentate (p = cper/cret). Since the β-lactoglobulin is small in comparison to the nominal pore size, a permeation p = 1 could principally be expected.

Low ionic strength

High ionic strength Stern layer

Medium ionic strength

Diffuse layer Distance/thickness

1 κ

Fig. 1.2 Electrical double layer around a charged colloidal particle as a function of ionic strength. At higher levels of ionic concentration in the outer phase, the charge of particles is shielded at a closer distance from the surface such that the hydrodynamic radius is reduced.

cing separation results, factors that are less specific and not fully predictable when compared with other separation techniques. Nowadays, membrane separations are often combined with other technologies such as centrifugal or chromatographic separations, in hybrid processing modes.

6

Dairy-derived ingredients

1.2 1.2.1

State-of-the-art separation techniques

Interfacial effects in the separation of dairy components by means of membrane techniques Membrane separation has become a widely-used unit operation in dairy products manufacture. The concentration of proteins from whey in the manufacture of so-called WPC products has been introduced during the early 1970s and has since been established also in speciality operations. Ultrafiltration is the most frequently used membrane process, mostly run in the crossflow processing mode. In contrast to dead-end filtration as it is applied in sterile filtration of biological media or in cake filtration in the beverage industry, the product to be concentrated is passed along the membrane surface to induce friction and, thus, a wall shear stress at the membrane surface. It is desirable to remove deposited particles from the surface of the membranes in order to avoid an overly strong effect of the deposited layer on permeation and retention. Therefore, the volume stream or fluid velocity is of great effect, as shown in an example in Fig. 1.3. It can be seen that the permeation increases linearly as a function of the transmembrane pressure difference when water is flowing across and permeating through the membrane. The permeation of protein solutions, on the other hand, is increasing less steeply and under-proportionally as the pressure goes up. The flux is determined by the membrane only at low transmembrane pressure differences (∆pTM). Above approximately ∆pTM = 3 bar, depending on flow rate, the flux values flatten off. Beyond this point, often referred to as critical flux or critical ∆pTM, the permeation is controlled by a compressed, dense layer of deposited protein particles. These data demonstrate that, despite high wall shear stresses, the aim of preventing deposit formation is not fully reached and the deposit layer acts in fact as a secondary membrane which may even dominate the separation process. The question arises in this context why the deposition of particles cannot be fully prevented. The answer lies in a dilemma situation of crossflow membrane filtration: ∆pTM and wall shear stress τW are dependent factors: the higher the wall shear stress (exerted by the volume flow along the membrane), the higher is the static pressure on the retentate side of the membrane. This, in turn, induces a high level of convective flow of solution towards the membrane as result of the transmembrane pressure ∆pTM. The retained particles accumulate at the membrane surface, thus generating a concentration increase in comparison to the bulk solution. This particularly applies to the region at the front end of the membrane, despite the fact that diffusional backflow and wall shear stress partially reduce the excess in concentration of retained particles. Further to that, the pressure loss along a flow path will create an inconsistent transmembrane pressure pattern along the membrane surface. These factors, in combination, impair the full control of processing conditions such that the membrane as a selective barrier is fully responsible for retention and permeation.

Novel approaches for the separation of dairy components

7

100

90

Milk protein solutions CPr = 4.5% θ = 30 °C

80

H2O

Flux (kg m–2 h–1)

70 τW (N/m2)

60

34.8 ∆pD

50

26.0 40 18.7 30 11.8 20 5.9 10 2.2 0 0

1

2

3

4

5

6

7

∆pTM (bar)

Fig. 1.3 Effect of transmembrane pressure difference ∆pTM and wall shear stress τW at a membrane surface on permeation rate in the ultrafiltration of milk protein solutions. The membrane material was poly sulfone (PS), nominal cut-off 25 kD.

An additional explanation for the difficulty of avoiding deposit formation is that dairy proteins, in particular casein micelles, are too large to be able to diffuse back from the membrane into the bulk solution and too small to be affected by lift forces imposed by the fluid stream. Calculations by Altmann and Ripperger1 led to results presented in Fig. 1.4 explaining this situation in more detail. The diagram depicts the flux rate as a function of the size of the particles present in an aqueous model phase, water in this case. The flux curve can be divided into two regions: (i) at small particle sizes, the removal of deposited particles from the membrane surface is influenced by diffusion as a mechanism for particle removal. The smaller the dispersed particle, the higher the flux; (ii) the right wing of the graph, with an inverse slope as compared to the left side of the graph, can be

8

Dairy-derived ingredients 10000

Flux (kg m–2 h–1)

Region dominated by hydrodynamic forces 1000

Region dominated by diffusion

100

10 10

100 1000 Particle size (nm)

10000

Fig. 1.4 Permeation rate as a function of particle size in model suspensions of Al2O3, TiO2, SiO2 and Kieselgur particles in sizes between 23–13 200 nm. The material, however, in this case is not of importance in relation to the size.1

explained by the fact that in this region, particles are affected by the shear force exerted by the fluid stream across the membrane surface. The larger the particles, the more effective they are influenced by hydrodynamic lift forces. It can be seen that the filtration of particles around 200 nm leads to a minimum of the curve. Neither diffusion nor lift forces are significantly affecting the particles deposited at the surface in this region. Considering these results, obtained for a model system consisting of inorganic particles in water, one can gain a basic understanding of why the deposition of casein micelles in milk, whose average size is in the area of 200 nm, can hardly be prevented in the cross flow filtration mode. Nevertheless, separation of casein micelles and native whey proteins has been investigated and successfully applied during recent years to obtain both protein fractions, without precipitation of either of them. This allows exploitation of their individual functional properties or shifting the ratio of both protein groups in order to influence the resulting textural profile of gels after acid or thermal coagulation. A simplified processing scheme is shown as an insert in Fig. 1.5a. Skim milk is passed through a microfiltration unit equipped with a ceramic membrane (SCT membrane ZrO2/Al2O3; nominal pore size 0.1 µm). The MF-permeate containing the whey proteins is then concentrated by an ultrafiltration unit concentrating the whey proteins. The UF-permeate containing the soluble low molecular weight substances of milk (lactose, minerals) at the original concentration, in turn, is recirculated to the feed tank

Novel approaches for the separation of dairy components

9

(a) 600:1 Microfiltration Skim milk θ = 55 °C ∆pM = 40 kPa τW = 150 Pa

Casein/Whey protein ratio

500:1 400:1

Skim milk

300:1

MF-permeate

MF/Diafiltration Casein micelles

200:1

Ultrafiltration UF permeate Whey proteins

100:1 0

0

1

2

3 4 Diafiltration steps

5

6

(b)

50% –1

In (Ct/C0)

20% –2 10%

–3 5%

–4

Amount of whey protein to be washed out

100%

0

3% 0

1

2

3

4

5

6

Diafiltration step

Fig. 1.5 Casein–whey protein ratio as a function of diafiltration steps in the fractionation of caseins and whey proteins by microfiltration from milk: (a) linear scale; (b) Logarithmic scale. The MF membrane used was a SCT membrane made of ZrO2/Al2O3; the diafiltration was conducted using UF permeate from milk.

10

Dairy-derived ingredients

of the MF-unit to wash out the remaining whey proteins in a diafiltration mode. Using the UF permeate instead of water as a medium for diafiltration allows one to avoid a change in the native aqueous environment of the casein micelles. The number of volume turnovers to fully achieve a complete separation of the milk protein main fractions depends on the degree of permeation of the whey proteins through the MF-membrane. This is why the expected separation effect has to be defined in the form of the permeation value p, which is the ratio of this component in the retentate c0 and in the permeate c: p = c / c0

[1.1]

Theoretically, i.e. assuming unlimited permeation, the permeate concentration of substances passing through is equally as high as in the retentate, i.e. c = c0 and p = 1. Since pressure-driven membrane processes principally separate according to molecule size relative to the pore size of the membrane, the separation efficiency is driven by the amount of filtrate, which convectively carries the permeating component through the membrane pores. Figure 1.5 further demonstrates an experimental result from a fractionation process of casein micelles (medium size 150 nm) from whey proteins (medium size 3–4 nm) by means of microfiltration (SCT membrane ZrO2/ Al2O3, nominal cut-off: 0.1 µm). It is obvious that, despite optimal choice of the theoretically best membrane pore size, a diafiltration process is required to wash out the whey proteins completely. The number of diafiltration steps actually required depends on the real permeation value p, which is lower than the theoretical value of p = 1 due to unspecific retention effects, and is depicted as the x-axis, while the y-axis shows the casein–whey protein ratio, starting from approximately 8 : 2 (close to the origin), which is the original ratio of casein and whey proteins in milk. As can be seen from Fig. 1.5, five to six diafiltration steps are required to reduce the content of whey proteins in the casein retentate by 95%, based on a permeation value p = 0.5, which is considerably lower than the theoretically assumed value of p = 1. This can be easily explained by the deposit formation of the retained casein micelles, as well as by an unspecific adsorption of whey proteins at the membrane surface. These observations confirm that we need to improve the understanding of the boundary layer effects at the membrane surface in order to be able to minimize their impact.

1.2.2 Separation of dairy components by means of chromatography In contrast to its widespread use in the sugar industry, chromatography is a separation technique less common in the dairy industry. The use of chromatographic techniques for the fractionation of whey proteins, however, has offered new ways for the extraction of highly effective protein

Novel approaches for the separation of dairy components

11

components of a natural milk source, which are products with an important role in promoting human health.2 Together with the main fractions, β-lactoglobulin and α-lactalbumin, present in whey protein isolate often used in infant formula, immunoglobulins are also important food supplements. Glycomacropeptide plays a growing role as a protein source in phenylalanine-free food for phenyl-ketonuria patients. These are just a few examples indicating that the functional properties of individual protein fractions are appreciated more and more in the development of food systems with improved structures or with bio-functional properties. Chromatography has been continuously developed over the last twenty years to remove lactose from milk, to serve a latently existing market for products for lactose-intolerant consumers, across Europe and in developing dairy markets in Asia.3 Meanwhile, the principle of chromatographic separation is being combined with the possibilities of other separation processes to improve throughput and to optimize the ratio of throughput and specificity, as will be shown below. 1.2.3 Separation of dairy components by centrifugal force Beyond the standard separation of fat and skim milk, centrifugation as a unit operation is applied, in combination with membrane technology, to produce precipitated calcium salts from acid milk permeate.4 According to an industrialized process, UF-permeates of whey or milk are concentrated by reverse osmosis to approximately 18 to 20% total solids. The pH is then adjusted to alkaline and the solution heated to promote the precipitation of calcium phosphate. While the reaction occurs instantaneously, a short holding time is applied to stabilize the precipitate, which subsequently is separated from the lactose solution either by centrifugation or cross-flow membrane filtration. The lactose stream is available for further downstream processing. The calcium phosphate slurry is further refined and concentrated by ultrafiltration with diafiltration to the desired end composition and lactose recovery rate.

1.3 1.3.1

Process intensification by hybrid processes

Thermal separation of whey proteins in combination with membranes As mentioned above, chromatography can be applied on a large scale to separate proteins. Nevertheless, chromatography may be limited in terms of throughput, and also with regard to the cleaning process, which requires high amounts of chemicals to restore suitable conditions for the next production cycle. Therefore, alternative processes have been proposed to enable the separation of two originally equally-sized molecules by membrane separation, whereby the protein mixture is subjected to a thermal process under conditions, such as specific pH, ionic strength, protein and

12

Dairy-derived ingredients

lactose concentration, which selectively affect individual proteins leading to their aggregation, while the target protein remains unaffected and native in solution.5 The aggregated protein can then be easily removed from the solution by microfiltration. Figure 1.6 provides an example demonstrating this concept by an assessment of the reaction rate constant of the thermal aggregation process. The system heated is whey, i.e. comprised of β-lactoglobulin and α-lactalbumin, the two main whey proteins in the natural aqueous phase of milk. It can be seen by comparing the reaction rate constants, a measure for the rate of the aggregation, that the proteins react differently, depending on lactose and protein contents. β-Lactoglobulin, for instance, reacts relatively fast at low lactose and low protein contents, while α-lactalbumin reacts more slowly under the same conditions. Other regions on these graphs can be identified where the opposite is the case, or where both proteins are fast in aggregating. From these fundamental data, and from other results related to other thermal and milieu conditions, optimized thermal processes can be derived which allow one of the proteins to be converted into aggregates while the respective other protein remains native. Therefore, microfiltration can be applied to remove the aggregated protein as a so-called microparticulate (which has its own technological value), while the native protein can be obtained from the microfiltration permeate by ultrafiltration. Figure 1.7 depicts two examples related to this selective whey protein separation process.

1.3.2

Enzyme-induced crosslinking and separation of dairy proteins or peptides Another example of the combination of other unit operations with membrane filtration is the selective upstream treatment of caseinomacropeptide (CMP) from sweet whey. The presence of CMP may sometimes be undesired; for instance, when a selective fractionation of whey proteins as described above is wanted. Tolkach and Kulozik6 developed an enzyme assisted crosslinking process of CMP using transglutaminase (TG). A condition for the crosslinking of proteins or peptides is the presence of two amino acids, namely lysine and glutamine, between which TG creates isopeptide bonds. Thus, crosslinking the small CMP molecule is induced, generating peptide aggregates large enough to be removed by microfiltration from whey, as shown in Figures 1.8 and 1.9. The remaining whey proteins can be further fractionated either using chromatography or the processing options presented in Fig. 1.7.

1.3.3

Membrane adsorption chromatography for the separation of proteins and peptides from whey As mentioned above, the permeation process in membrane technology is influenced by a variety of factors still not fully understood and, in addition,

Novel approaches for the separation of dairy components

13

Aggregation rate constant [(gL–1)1–nS–1]103

(a)

10 90°C 8 7 5 4 3 0

5 1 La

4 cto

2 se

3 co

3 nte nt

2 4 (%

1 5

)

0

in

te Pro

t (%

)

n nte

co

Aggregation rate constant [S–1]103

(b)

14 90°C

12 10 8 6 4 2 0 0.5

5.5 1.5

La

4.5 2.5 se 3.5 co nte 4.5 nt (% )

3.5

cto

nt nte

2.5 1.5 5.5

0.5

in

ote Pr

)

(%

co

Fig. 1.6 Reaction rate constant of aggregation for (a) β-lactoglobulin (following a reaction order of n = 1.5) and (b) α-lactalbumin (following a reaction order of n = 1) as a function of protein and lactose contents in the heating solution at 90 °C.5

14

Dairy-derived ingredients

(a) Whey

UF/diafiltration 3.5% Protein

Heat treatment 93°C/20 s; pH 7.5

pH adjustment

α-la

β-lg B

Native Heated

500 400 300 200 100

β-lg A

0 5

7.5

10

12.5

15

17.5

20 22.5 (min)

MF 0.1 µm

Retentate denatured β-Lg

Permeate native α-La

(b) Whey or WPC β-Lg B

UF-diafiltration 6% β-Lg, citrate

Heat treatment 56°C/40 min; pH 3.6 Microfiltration 0.1 µm

500 400 300 200 100 0

Native Heated α-La

5 Retentate denatured α-La

Permeate native β-Lg

RO of UFpermeate

Diafiltration (UF)

β-Lg A

7.5

10

12.5

15

17.5

20 22.5 (min)

Fig. 1.7 Process diagrams for the separation of (a) β-lactoglobulin or (b) α-lactalbumin from pre-treated whey. Also shown are the respective chromatograms demonstrating that the native protein has hardly been affected by the upstream treatment. The dotted chromatogram lines show the whey protein composition prior to heat treatment, the solid lines the chromatograms after heat treatment.5

Novel approaches for the separation of dairy components

15

Remaining CMP (%)

100 WPC cpr = 6% (TG): 3 U/g protein θ = 40°C

80 60 40

Untreated

20 0

CMP A

10 min 120 min

0

20 40 60 Incubation time (min)

80

Glycosylated CMP

CMP B

Fig. 1.8 Enzymatic crosslinking of caseinomacropeptide and removal from whey by microfiltration: Kinetics of CMP aggregation and whey protein HPLC chromatograms highlighting the loss of CMP over time. Both the glycosylated and the non-glycosylated parts of CMP are crosslinked and are therefore reduced in the chromatogram since they no longer remain soluble.

WPC

Treatment with transglutaminase UF permeate MF, 0.14 µm

UF, 25 kDa MF permeate

Crosslinked CMP and remaining casein curd

WPC w/o CMP

MF retentate

UF retentate

Fig. 1.9

Fractionation of whey proteins

Block diagram of a process using enzymatic crosslinking to aggregate CMP and microfiltration to remove the aggregates.

which are relatively unspecific. This is why separation tasks are often achieved in a more targeted way by chromatographic techniques. These techniques, however, are often limited by the low velocity of the diffusional processes which are responsible for the transport of substances into and out

16

Dairy-derived ingredients

of the chromatographic beads. Therefore, novel hybrid systems have been developed combining the features of both processes, namely specificity of ligands in chromatography (quaternary ammonium ions in this case) and high throughput of membranes. Such systems are called membrane adsorption chromatography units (MAC), which consist of natural polymers, such as cellulose, as carrier material; these can be easily modified at their surfaces. Similar to adsorber beads, the surfaces can carry charged or otherwise adsorptive ligands (in this study, again using quaternary ammonium ions), which are convectively brought in contact with the substrates in the filtrate stream passing along them. Therefore, diffusional processes are much less limiting as compared to conventional chromatographic systems. As could be shown in a study for the separation of fractions of caseinomacropetide (CMP), conventional ion exchange (IEX) chromatography and MAC can successfully be applied to separate the glycosylated from the unglycosylated parts, based on the fact that the glycosylated peptides are negatively charged. This might be of practical importance because of certain bioactivities and specific technological functionalities of CMP.7 Without the MAC technique, this separation would be only possible by standard ion exchange chromatography (IEX). Figure 1.10 compares the separation results of MAC and conventional ion exchange chromatography. The diagrams depicts the elution curves detected by a UV detector as a function of elution time. As can be seen, the separation effect is very similar between

0.0 2500

0.5

1.0

1.5

2.0

Time (min) MAC Adsorber

UV absorption 218 nm

2000

1500

1000

IEX-column

500

0 0

2

4 6 Time (min) IEX column

8

Fig. 1.10 Elution curves from a separation process of aglyco- and glyco-CMP from whey using ion exchange chromatography in comparison to membrane adsorption chromatography, both carrying quaternary ammonium groups as ligands.8

Novel approaches for the separation of dairy components

17

the systems. They both achieve a base line separation of peaks. The IEX chromatogram indicates that the glyco-CMP consists of various subfractions (see also Fig. 1.8) which could be further separated. However, the target here was to separate only the two bulk fractions, glyco- and aglyco-CMP. More importantly, it was shown that, in fact, MAC achieves the separation effect in a quarter of the time required by conventional IEX chromatography. It can be predicted that MAC technology will have further applications in the food industry or in pharmaceutical applications, since different ligands can be linked with the carrier surfaces and that a wide spectrum of separation tasks could successfully be solved by using MAC units.

1.4

Alternative membrane processing concepts

At the beginning of membrane technology, only polymeric materials were available and they are still dominating the market. Later on, ceramic or other inorganic materials have been introduced, which allow more extreme conditions of operation, cleaning and sterilization and have, partially, also more specific separation capabilities, and therefore have occupied a significant part of the membrane market. In addition, innovative technical solutions have been developed allowing a better control of processing conditions and, thus, of separation results, as will be shown below. 1.4.1

Membrane filtration with uniform transmembrane pressure difference As was shown above, high wall-shear stresses in combination with subcritical and uniform transmembranes pressure differences should yield better separation results. Different approaches have been taken to achieve improved situations with a better deposit control. The first system was developed during the 1970s, achieving a uniform transmembrane pressure difference ∆pTM by means of pumping the permeate in a circular mode through the permeate side of the membrane modules.9 The volume flow through the permeate side of the module can be adjusted such that the pressure loss is equivalent to the pressure loss along the retentate side of the membrane. By doing so, the pressure difference at each point along the membrane is the same, i.e. uniform. This results in a processing mode where, in contrast to conventional crossflow systems, volume flow rate and transmembrane pressure are no longer dependent factors delivering the same ∆pTM. This, in turn, allows the control of deposit formation through high levels of wall shear stress without simultaneously creating highly convective transportation of deposit-forming material towards the membrane surface. A processing scheme, as well as the related pressure situations on the retentate and permeate sides, is schematically depicted in Fig. 1.11 according to Sandblom.9

18

Dairy-derived ingredients Product

0 100 200 300 400 kPa

Retentate PI

PI

Permeate Permeate UTP

PI

PI

Membrane Permeate

Retentate

0 100 200 300 400 kPa

Fig. 1.11 Uniform Transmembrane Pressure (UTP) mode membrane filtration.9 The pressure gradient on the retentate side of the membrane is equal to an induced pressure loss on the permeate side, thus creating the same transmembrane pressure along the membrane.

A result from a microfiltration experiment fractionating milk proteins is shown in Fig. 1.12, comparing the UTP mode with the classical crossflow mode. The permeation rate is strongly time-dependent in the case of crossflow MF, while for the UTP mode a much higher and constant flux can be obtained, indicating that the control of deposition of the retained casein micelles was more effective.

1.4.2 Gradient membranes The positive effect described above stood behind the development of socalled gradient membranes made from ceramic materials, which have inbuilt gradients in permeation resistances. One of the concepts is based on a gradient in porosity within the membrane support layer (System Pall Exekia/France); another is based on the gradient in thickness of the selective front layer of the membrane (System TAMI/France). Either of the gradients can be established during membrane manufacture such that the permeation resistance along the flow path is inversely varied according to the linear decrease of static pressure on the retentate side. Provided that the respective gradient and the pressure loss on the retentate side are ideally compensating each other, the result is the same flux along the membrane. Figure 1.13 schematically depicts both gradient membrane concepts.

Novel approaches for the separation of dairy components

19

80 with UTP

flux J [I/(hm2)]

60

40 without UTP Microfiltration Skim milk θ = 55 °C ∆pTM = 40 kPa τW = 150 Pa

20

0

0

15

30 Time (min)

45

60

Fig. 1.12 Permeation rate in microfiltration-based fractionation of skim milk comparing crossflow and UTP modes using ceramic tubular membranes (ZrO2/Al2O3).10 (a)

(b) Flux

Membrane support with porosity gradient

Flux

Selective layer Product stream

Support layer Product stream

Selective layer with a gradient in thickness

Fig. 1.13 Schematic depiction of membranes (a) with a gradient in membrane support porosity or (b) in the thickness of the selective layer at the membrane surface.

20

Dairy-derived ingredients

Gradient membranes should have the same effect (i.e. isoflux along the membrane) as the UTP processing mode, provided that the fluid properties (viscosity, concentration), which are varying according to the concentration through the stages of a larger membrane plant and which are responsible for the pressure loss, are compatible with the gradient of the respective membranes confronted with these variable properties of the retentate stream.

1.4.3 Dynamic membrane systems Another approach in creating wall shear stress has been realized by the development of dynamic membrane systems with either rotating or oscillating membranes. The movement of the membrane, instead of high volume throughputs of the product, allows one to disentagle pressure difference and wall shear stress similar to the systems described above. One of the first systems applying this concept has been the simple stirred vessel membrane cell, mainly for laboratory scale applications. A truly dynamic membrane system, the first of its kind, was the rotor–stator system by Sulzer (Winterthur/Switzerland) with membrane equipped cylindrical rotors and stators creating a circular gap between them, shear forces and Taylor vortices producing the required wall shear stress and removing deposited material. This concept has been later transferred to the development of flat sheet circular membrane systems, and deposit removal or control could be realized without large volume streams passing along the membrane surfaces. Figure 1.14 schematically shows one of the presently available systems with overlapping, rotating circular membranes. Figure 1.15 depicts results from a microfiltration experiment using an oscillating membrane system (PallSep; Pall/NJ) for the fractionation of milk proteins, i.e. whey proteins from caseins, with variations in the amplitude of the oscillation. As can be seen, the permeation is strongly dependent on the transmembrane pressure difference, despite the fact that a low volume stream is applied. This clearly shows that the system is operated in a

Permeate flow Product flow Retentate flow

Fig. 1.14

Scheme of a dynamic membrane system with rotating membranes.

Novel approaches for the separation of dairy components

21

(a) 70 Amplitude of oscillation

Permeate flux (kg m–2 h–1)

60

7 mm

50 4.5 mm 40

30 3 mm 20

10

0 0

0.2

0.4

0.6

0.8

1

Transmembrane pressure ∆pTM (bar) (b) Specific mass flow β-Lg (g m–2 h–1)

2000

Amplitude of oscillation

1800

7 mm

1600

4.5 mm

1400 1200 1000

3 mm

800 600 400 200 0

0

0.2

0.4

0.6

0.8

1

Transmembrane pressure ∆pTM (bar)

Fig. 1.15 (a) Permeation rate and (b) protein mass flow in MF using an oscillating membrane system for the fractionation of milk proteins.

membrane controlled region, where the deposit formation is negligible and therefore does not influence the permeation in a major way. On the other hand, the permeation is controlled by the deposited layer of retained protein particles at higher levels of the transmembrane pressure difference,

22

Dairy-derived ingredients

in particular when the amplitude of oscillation is low. Figure 1.15b depicts the mass flow of smaller proteins, using β-lactoglobulin as an example, showing that an undesired retention can obviously be prevented as long as the critical value for ∆pTM is not exceeded.

1.4.4 Electric field assisted membrane techniques Electrodialysis has long been applied in the dairy industry to separate ionic components, mainly from whey, using ion exchange membranes. Alternatively, charged particles can be removed from solutions using other kinds of membranes assisted by an electrical field, as has been demonstrated by Weigert et al.11 The authors made use of the effect of an electrical field to enhance the effect of fluid dynamical lift forces in removing charged particles from membrane surfaces. The electrical field was polarized according to the net particle charge such that the direction of the forces was pointing away from the membrane surface. The results, depicted in Fig. 1.16, clearly show that submicron particles in a model system with anorganic (crystobalite) particles were effectively forced away from the membrane surface yielding a significantly higher flux rate as compared to the same filtration without an electrical field. Although these results were obtained for a non-food system, the potential of this principle for more complex solutions in dairy or other food applications exists and should be of interest to validate.

1500

Permate flux [kg m–2 h–1]

150 V/cm 1200

900

600

300

0 V/cm

0 0

5

10 Time [h]

15

20

Fig. 1.16 Effect of an electrical field on the permate mass flow in crossflow microfiltration of anorganic crystobalite particles in comparison to conventional crossflow filtration.11

Novel approaches for the separation of dairy components

1.5

23

References

1 j. altmann and s. ripperger (1997): Particle deposition and layer formation at the crossflow microfiltration. J. Membrane Sci., 124, 119–128 2 w. strohmaier (2003): Chromatographic fractionation of whey proteins. Proceedings, IDF Conference on Advances in Fractionation and Separation: Processes for novel dairy applications, Bruges, Belgium 7–12 Sept., pp. 81–93 3 p. jelen and o. tossavainen (2003): Low lactose and lactose free milk and dairy products – prospects, technologies and applications. Australian J. Dairy Technol. 58 (2), 161–165 4 c. f. brothersen, n. f. olson, and t. richardson (1982): Recovery of calcium phosphate from ultrafiltration permeates. J. Dairy Sci. 65, 17–23 5 a. tolkach and u. kulozik (2005): Optimization of thermal pre-treatment conditions for the separation of native α-lactalbumin from whey protein concentrates by means of selective denaturation of β-lactoglobulin. J. Food Sci., 70 (9) 557–566 6 a. tolkach and u. kulozik (2005): Fractionation of whey proteins and caseinomacropeptide by means of enzymatic crosslinking and membrane separation techniques. J. Food Eng., 67 (1–2) 13–20 7 c. thomae, i. krause, and u. kulozik (2006): Precipitation behaviour of caseinomacropeptides and their simultaneous determination with whey proteins by RP-HPLC. Int. Dairy J. 16 (4) 285–293 9 r. m. sandblom, US Pat. 4 105 547, 1978 10 u. kulozik and m. kersten (2001): Fraktionieren von Proteinen mittels Mikrofiltration. Chemie Ingenieur Technik 73, 1622–1625 11 t. weigert, j. altmann, and s. ripperger (1999): Crossflow electrofiltration in pilot scale. J. Membrane Sci., 159, 253–262

2 Understanding the factors affecting spray-dried dairy powder properties and behavior P. Schuck, INRA, France

Abstract: In view of the increased development of filtration processes, the dairy industry requires greater understanding of the effects of spray drying on the quality of protein powders. Spray drying parameters have an important role in the quality of dairy powders during drying, storage and rehydration. The residence time of the droplet, and then of the powder, is so short that it is very difficult to study the mechanism of the structural change in the protein without fundamental research into relationships with the process/product interactions. Following an introduction to spray drying, this chapter covers four areas, i.e. the principles of spray drying, the properties and qualities of spray-dried dairy powders, the effects of spray drying parameters on powder properties, and the control and improvement of powder properties. Key words: spray drying, powders, dairy products, physical properties, rehydration.

2.1

Introduction

The purpose of the dehydration of milk and derivates is to stabilize these products for their storage and later use. Milk and whey powders are used mostly in animal feeds. With changes in agricultural policies (such as the implementation of the quota system and the dissolution of the price support system in the European Union), the dairy industry has been forced to look for better uses for dairy surplus and for the by-products of cheese (whey) produced from milk, and buttermilk produced from cream. Study of the reuse of protein fractions in terms of nutritional qualities and functionality has led us to believe that they could have several applications. In the past 30 years, the dairy industry has developed new technological processes for extracting and purifying proteins (e.g. casein, caseinates, whey

Understanding the factors affecting spray-dried dairy powder

25

proteins, etc.) such as milk protein concentrate (MPC), milk protein isolate (MPI), whey protein concentrate (WPC), whey protein isolate (WPI), micellar casein concentrates (MCC) and isolates (MCI), whey concentrates, and selectively demineralized whey concentrates, mainly because of the emergence of filtration technology (e.g. microfiltration, ultrafiltration, nanofiltration and reverse osmosis). This recent emergence of new membrane separation techniques and improvements in chromatographic resins now provide the dairy technologist with several types of technique for the extraction and purification of almost all of the main milk proteins. (Maubois, 1991; Fauquant et al., 1988; Pierre et al., 1992, Schuck et al., 1994a,b; Jeantet et al., 1996). The most frequently used technique for the dehydration of dairy products is spray drying. It became popular in the dairy industry in the 1970s, but at that time there were few scientific or technical studies on spray drying and, in particular, none on the effects of spray drying parameters or on the effects of the physico-chemical composition and microbiology of the concentrates on powder quality. Manufacturers acquired expertise in milk drying, and eventually in whey drying, processes through trial and error. Because of the variety and complexity of the mixes to be dried, a more rigorous method based on physico-chemical and thermodynamic properties has become necessary. Greater understanding of the biochemical properties of milk products before drying, water transfer during spray drying, the properties of powders and influencing factors is now essential for the production of milk powder. The lack of technical and economic information and of understanding of scientific methods prevents the manufacturer from optimizing his plant in terms of energy costs and powder quality. A dairy powder is characterized not only by its composition (proteins, carbohydrates, fats, minerals and water) but also by its microbiological and physical properties (bulk and particle density, instant characteristics, flowability, floodability, hygroscopicity, degree of caking, whey protein nitrogen index, thermostability, insolubility index, dispersibility index, wettability index, sinkability index, rehydration time, free fat, occluded air, interstitial air, particle size, water activity, glass transition temperature, etc.) which form the basic elements of quality specifications, and there are well-defined test methods for their determination according to international standards (Pisecky, 1986, 1990, 1997; American Dairy Products Institute, 1990; Masters, 2002). These characteristics depend on drying parameters (type of tower spray drier, nozzles/wheels, pressure, agglomeration and thermodynamic conditions of the air, such as temperature, relative humidity and velocity), the characteristics of the concentrate before spraying (composition/physico-chemical characteristics, viscosity, thermo-sensitivity and availability of water) and storage conditions. Several scientific papers have been published on the effects of technological parameters on these properties (Baldwin et al., 1980; Pisecky, 1980, 1981, 1986; De Vilder, 1986; Tuohy, 1989; Jeantet et al., 2008a; Masters, 2002) (Fig. 2.1). Water content, water

26

Dairy-derived ingredients Physical properties Concentrate properties

Water availability

Drying conditions

Storage conditions

Fig. 2.1

Biochemical properties Powder quality

Rehydration properties

Microbiological qualities

Properties and qualities of powders.

dynamics and water availability are among the most important properties for all these powder properties and powder characteristics. The aim of this chapter is to provide information on four areas, i.e. principles of spray drying, properties and qualities of spray-dried dairy powders, effects of spray drying parameters on powder properties, and control and improvement of powder properties.

2.2

Principles of spray drying

Drying is defined as the removal of a liquid (usually water) from a product by evaporation, leaving the solids in an essentially dry state. A number of different drying processes are in use in the dairy, food, chemical and pharmaceutical industries, such as spray-drying, fluid bed drying, roller drying, freeze-drying, microwave drying and superheated steam drying. Due to considerations related to drying economics and final product quality, the only processes of significance in milk protein powder manufacture are spray-drying and fluid bed drying (most often in combination). Only these two combined drying processes will be discussed in this section. The basic principle of spray-drying is the exposure of a fine dispersion of droplets, created by means of atomization of preconcentrated milk products, to a hot air stream. According to Pisecky (1997), spray drying is an industrial process for the dehydration of a liquid by transforming the liquid into a spray of small droplets and exposing these droplets to a flow of hot air. The very large surface area of the spray droplets causes evaporation of the water to take place very quickly, converting the droplets into dry powder particles. The small droplet size created, and hence large total surface area, results in very rapid evaporation of water at a relatively low temperature, whereby heat damage to the product is minimized. (Refstrup,

Understanding the factors affecting spray-dried dairy powder

27

2003) In fact, when a wet droplet is exposed to hot dry gas, variations in the temperature and the partial pressure of water vapor are spontaneously established between the droplet and the air: • Heat transfer from the air to the droplet occurs under the influence of the temperature gradient; • Water transfer occurs in the opposite direction, explained by variation in the partial pressure of water vapor between the air and the droplet surface. Air is thus used both for fluid heating and as a carrier gas for the removal of water. The air enters the spray drier hot and dry, and leaves it wet and cool. Spray drying is a phenomenon of surface water evaporation maintained by the movement of capillary water from the interior to the surface of the droplet. As long as the average moisture is sufficient to feed the surface regularly, the evaporation rate is constant. If not, it decreases. The drying kinetics are related to three factors: (i)

The evaporation surface created by the diameter of the particles. Spraying increases the exchange surface (1 L of liquid sprayed in particles of 100 µm diameter develops a surface area of 60 m2, whereas the surface area for one sphere of the same volume is approximately only 0.05 m2). (ii) The difference in the partial pressure of water vapor between the particle and the drying air. A decrease in the absolute humidity of the air and/or an increase in the air temperature tends to increase the difference in the partial pressure of water vapor between the particle and the drying air. (iii) The rate of water migration from the center of the particle towards its surface. This parameter is essential for the quality of dairy powders. Indeed, it is important that there is always water on the surface of the product so that the powder surface remains at the wet bulb temperature for as long as possible. The rate of water migration depends on the water diffusion coefficient, which varies according to the biochemical composition, water content and droplet temperature. This is why calculation of this coefficient is complex and the mathematical models available are not easily exploitable by the dairy industry. To define the components of a spray drying installation according to Masters (1991), Pisecky (1997) and Westergaard (2003), the main components of the spray drier shown in Fig. 2.2 are as follows: • Drying chamber (Fig. 2.2, No. 7). • Air filtration (Fig. 2.2, No. 17). • Air heating system. The drying air can be heated in various ways: either indirectly by steam, oil, gas or hot oil, or directly by gas or electricity (Fig. 2.2, No. 5).

28

Dairy-derived ingredients 4 17

5

Outlet air

Inlet air 15 3 2

10

9

8 7

Concentrate 11

1

12

16 16

15 Inlet air

16 17

4

5 14 17 13

5

Inlet air

Powder Inlet air 17 13

6

Fig. 2.2 Multiple effect spray-dryer; 1 Feed pump; 2 Feed flow; 3 Sprayer/Air disperser; 4 Inlet air fan; 5 Air heater; 6 Air cooler; 7 Drying chamber; 8 Primary cyclone; 9 Secondary cyclone; 10 Outlet air fan; 11 Integrated fluid bed; 12 Pressure conveyer system; 13 Vibro-fluidizer air fan; 14 Vibro-fluidizer; 15 Reincorporating fines; 16 Rotary valve; 17 Air filtration.

• Air distribution. The air flow chamber can be in co-current, countercurrent or mixed mode. The system of air distribution is one of the most vital points in a spray dryer. There are various systems, depending on the plant design and the type of product to be produced. The most common system is where the air disperser is situated on top of the dryer ceiling and the atomizing device is placed in the middle of the air disperser, thus ensuring optimal mixing of the air and the atomized droplets. • An atomizing device with a feed supply system such as a feed tank, feed pump, water tank, concentrate heater and atomizing device. The aim of atomizing the concentrate is to provide a very large surface area from which the evaporation of water can take place: the smaller the droplets, the greater the surface area; and the easier the evaporation, the greater the thermal efficiency obtained. The ideal situation from a drying point of view is a spray of droplets of the same size, where the drying time for all

Understanding the factors affecting spray-dried dairy powder

29

particles would be the same to obtain equal moisture content. As mentioned previously, air distribution and atomization are key factors in the successful utilization of a spray-dryer. Atomization is directly responsible for many distinctive advantages offered by spray-drying: first, the very short drying time of the particles; second, the very short particle retention time in the hot atmosphere and low particle temperature (wet bulb temperature); and finally, the transformation of the liquid feed into a powder with long storage stability, ready for packing and transport. In summary, the prime functions of atomization are: • to produce a high surface-to-mass ratio, resulting in a high evaporation rate • to produce particles of the desired shape, size and density. There are three types of atomizing device: nozzle atomizer (pressure, pneumatic or sonic), rotary atomizer (wheel or disc) and combined atomizer (rotary and pneumatic) (Fig. 2.2, No. 3). The basic function of pressure nozzles is to convert the pressure energy supplied by the high-pressure pump into kinetic energy in the form of a thin film, the stability of which is determined by the properties of the liquid, such as viscosity, surface tension, density and quantity per unit of time, and by the medium into which the liquid is sprayed. The basic function of a pneumatic nozzle, also called a two-fluid nozzle, is to atomize the feed by high air velocity The energy available for atomization in two-fluid atomizers is independent of liquid flow and pressure. The necessary energy (kinetic) is supplied by compressed air. Two-fluid atomization is the only successful nozzle method for producing very small particles, especially from highly viscous liquids. It is not normally used in the drying of milk products. In rotary atomizers, the liquid is accelerated continuously to the wheel edge by centrifugal forces produced by the rotation of the wheel. The liquid is distributed centrally and then extends over the wheel surface in a thin sheet and is discharged at high speed at the periphery of the wheel. The degree of atomization depends on peripheral speed, the properties of the liquid, and feed rate. According to Westergaard (2003), to select an optimal atomizer wheel, the liquid feed rate, peripheral speed and viscosity of the liquid should be taken into consideration. • Powder recovery system. Separation of the dried product can be achieved by a primary discharge from the drying chamber followed by a secondary discharge from a particle collector (using a cyclone, bag filter or electrostatic precipitation), followed by total discharge from the particle collector and finishing with final exhaust air cleaning in a wet scrubber and dry filter.

30

Dairy-derived ingredients Authorities normally conclude that a powder loss of 250 mg m−3 (obtained classically by using only cyclones) is too high, and set a standard of 50 mg m−3 (obtained by using cyclones plus bag filters or cyclones plus wet scrubber which requires a final cleaning of the air). For these reasons, i.e. authorities’ demand for reduced powder emission and powder producers’ demand for lower energy consumption and reduced space requirements, a new powder recovery system has been developed, the cleanable-in-place (CIP-able) bag filter, which replaces the cyclones/ bag filter (Westergaard, 2003).

According to Sougnez (1983), Masters (1991) and Pisecky (1997), the simplest types of installation are single-stage systems with a very short residence time (20–60 s). Thus, there is not enough residence time to obtain a real equilibrium between the relative humidity of the outlet air and water activity of the powder. The outlet temperature of the air must therefore be high, reducing the thermal efficiency of the single-stage spray drier. Generally, installations without any post-treatment system are suitable only for non-agglomerated powders not requiring cooling. If necessary, a pneumatic conveying system could be added to cool the powder while transporting the chamber fraction and the cyclone fraction to a single discharge point. The two-stage drying system consists of limiting the spray drying process to a process with a longer residence time (several minutes) to provide a better thermodynamic balance. This involves a considerable reduction in the outlet air temperature (to increase the powder moisture content), and also an increase in the inlet air temperature, and in the concentrate and powder flow rate without risk of thermal denaturation due to the increase in moisture content of the powder during the first stage (thermal protection). A second, final drying stage is necessary to optimize the moisture content, by using an integrated fluid bed (static) or an external fluid bed (vibrating), the air temperatures of which are 15–25°C lower than with a single-stage system to improve and/or preserve the quality of the dairy powder (Fig. 2.2, Nos 11 and 14). Consequently, the surrounding air temperature at the critical drying stage and the particle temperature are also correspondingly lower, thus contributing to further improvement in economics. The integrated fluid bed can be either circular (e.g. Multi Stage Drier (MSDTM) chamber) or annular (e.g. Compact Drier (CD) chamber). Two-stage drying has its limitations, but it can be applied to products such as skim milk, whole milk, pre-crystallized whey, caseinates, whey proteins and derivatives. The moisture content of the powder leaving the first stage is limited by the thermoplasticity of the wet powder, i.e. by its stickiness in relation to the water activity and the glass transition temperature (Roos, 2002). The moisture content must be close to 7–8, 9–10 and 2–3% for skim/ whole milk, caseinate/whey protein and pre-crystallized whey powders, respectively. The two-stage drying techniques can be applied to the production of both non-agglomerated and agglomerated powders, but this tech-

Understanding the factors affecting spray-dried dairy powder

31

nique is very suitable for the production of agglomerated powders, by separating the non-agglomerated particles from the agglomerates (i.e. collecting the cyclone fractions and re-introducing these fine fractions (called fines) into the wet zone around the atomizer of the chamber) (Fig. 2.2, No. 15). Three-stage drying systems, with an internal fluid bed as a second stage in combination with an external vibrating fluid bed as a third-stage drier, first appeared at the beginning of the 1980s and were called Compact Drier Instantization (CDI) or MSDTM. Today, they dominate the dairy powder industry (Fig. 2.2). Three-stage systems combine all the advantages of extended two-stage drying, using spray drying as the primary stage, fluid bed drying of a static fluid as the second drying stage and drying on an external vibrating fluid bed as the third drying stage. The final drying stage terminates with cooling to under the glass transition temperature. Evaporation performed at each stage can be optimized to achieve both gentle drying conditions and good thermal economy. CD or CDI is suitable for producing both non-agglomerated and agglomerated powders of practically any kind of dried dairy product. It can also cope successfully with whey powders, fat-filled milk and whey products as well as caseinates, both non-agglomerated and agglomerated. It has a fat content limit of about 50% fat in total solids. Powder quality and appearance are comparable with those of products from two-stage drying systems but they have considerably better flowability and the process is more economical. In comparison with CD, MSDTM can process an even wider range of products and can handle an even higher fat content. The main characteristic of MSDTM powder is due to very good agglomeration and mechanical stability, low particle size fractions (below 125 µm) and very good flowability. Optimization of the process allows considerable improvement in drying efficiency and the quality of the product obtained is generally better. The various advantages are: • improved thermal efficiency: significant reduction in outlet air temperature, permitting an increase in inlet air temperature; • reduced material obstruction: the capacity in one volume is two or three times greater than for a traditional unit; • considerable reduction in powder emission to the atmosphere: reduction of the drying air flow and increase in powder moisture content together decrease the loss of fine particles in the outlet air; • improved powder quality in relation to agglomeration level, solubility, dispersibility, wettability, particle size, density, etc. There are other examples of drying equipment such as the ‘tall form drier’, the ‘FiltermatTM drier’, the ‘ParaflashTM drier’, the ‘TixothermTM drier’ and the Integrated Filter DryerTM (IFDTM). All these towers have characteristics

32

Dairy-derived ingredients

related to the specific properties of the product being dried (e.g. high fat content, starch, maltodextrin, egg and hygroscopic products). In terms of energy balance, Westergaard (2004) showed that energy consumption varies according to the drying processes. Energy consumption to produce 1 kg of powder from skim milk concentrate at 45% of total solids is 1595, 1350, 1280, 1038 and 960 Kcal for a one-stage spray dryer, a twostage spray dryer, a two-stage spray dryer with high inlet air temperature, CDI and MSDTM, respectively. This is explained by the increase in the number of drying stages simultaneously increasing the residence time, allowing increase in the inlet air temperature, concentrate flow rate and finally energy yield while preserving/improving powder quality (Bimbenet et al., 2002; Jeantet et al., 2008b).

2.3

Properties and qualities of dairy powders

According to Caric´ (2003), the properties of milk powders are categorized as physical, functional, biochemical, microbiological and sensory. There is a significant interrelationship between them, which affects the final quality. The physical and functional properties of milk powders are especially important when the powders are intended for recombining and in the manufacture of various food products. When intended for use as a food ingredient, milk powders should be light in color, free from off-flavors and easy to hydrate, disperse and dissolve in water. The basic properties that determine the quality of milk powder, and where defects are most likely, include powder structure, solubility, water content, scorched particles, flowability, floodability, oxidative changes, flavor, color and micro-organism contamination.

2.3.1 Physical properties Powder structure The physical structure of a milk powder can be defined as the way in which its chemical components are distributed and connected. Powder structure is very strongly affected by the drying technique. Powder produced by roller-drying has a compact structure of irregular shape with no occluded air. Roller-dried powder particles have a low bulk density (300–500 kg.m−3), due to their irregular structure. The particles of spray-dried powder are spherical, with diameters in the range 10–250 µm. The particles contain occluded air and either large central vacuoles or smaller vacuoles which are distributed throughout the interior of the particles. The surface of spraydried skim milk powder particles is usually wrinkled but it is smooth for high protein powders. The high inlet air temperature and large temperature differential between the hot air and the powder particles are the main

Understanding the factors affecting spray-dried dairy powder

33

causes of wrinkle formation, as is also the presence of lactose (Caric´ and Kaláb, 1987; Mistry et al., 1992; Aguilar and Ziegler, 1993). Particle size distribution According to Carric´ (2003), the particle size of a powder, which affects its appearance, reconstitution and flow characteristics, depends mainly on the atomization conditions and the viscosity of the concentrate; high atomizing pressure and low concentrate viscosity reduce particle size. Powder density Densities are classified into three groups: bulk (apparent) density, particle density and the density of the dry milk solids; all three are very much interrelated. • Bulk density: Bulk density is regarded as the weight per unit volume and is expressed as kg.m−3. It is a very important property, both from the point of view of cost and market requirements. Bulk density is currently determined by measuring the volume of 100 g of powder in a 250 mL graduated glass cylinder. The bulk density of milk powders is a very complex property, being the result of many other properties and being influenced by a number of factors such as feed concentration, feed temperature, feed foamability, milk preheating, age thickening, feed composition, type of atomizer, particle temperature history and particle size distribution (Pisecky, 1997). Bulk density depends also on particle density and occluded and interstitial air. • Particle density: Particle density corresponds to the mass of particles (in grams) having a total volume of 1 cm3. Particle density is influenced mainly by the amount of entrapped air. The processing factors that contribute significantly to particle density are viscosity and the incorporation of air into the concentrate prior to drying. The type of spray atomization affects air retention. Certain types of centrifugal spraydried milk have more entrapped air than pressure spray products (Carric´, 2003). ° Occluded air: The occluded air content is defined and quantified by the difference between the volume of a given mass of particles and the volume of the same mass of air-free milk solids. Many factors influence the occluded air content in powder particles, including incorporation of air into the feed, the system chosen for spray drying the concentrate, whipping action before and/or during atomization, properties of the feed and the ability of the feed to form a stable foam. The content and state of proteins might markedly affect stable foam formation, while fat has the opposite effect. High-fat concentrates are much less susceptible to foaming than skim milk. Undenaturated whey proteins in skim milk have a greater tendency to foam, which can be reduced by heat treatment, which causes protein

34

Dairy-derived ingredients

denaturation. Concentrates with low total solid contents foam more than more highly concentrated materials. An increase in temperature from 10 to 40°C increases the tendency to form foam for skim milk concentrate at 40 and 45% of total solids related to decrease in viscosity. Therefore, an increase in temperature from 10 and 40°C reduces the tendency to form foam for concentrate at 50% of total solids. This result could be explained by a thermal denaturation (Pisecky, 1997; Carric´, 2003). ° Interstitial air: Interstitial air is defined as the difference between the volume of a given mass of particles and the volume of the same mass of tapped powder. This property depends primarily on the particle size distribution and the degree of agglomeration (Carric´, 2003). • Density of the dry milk solids: This density expresses the density of solids without any air and is given by the composition of the power (Pisecky, 1997; Westergaard, 2004). Flowability Flowability is the ability of a powder to flow freely, like sand, without forming lumps, clusters or aggregates. Flowability can be measured as the time (in seconds) necessary for a given volume of powder to leave a rotary drum through given slits (Haugaard Sorensen et al., 1978) or by the method developed by Carr (1965). Flowability depends also on particle size and shape, density and electrical charge. Large particles flow more easily than fines (particles with a diameter of 0.2 to < 0.6, sorption is dominated by the transformation of the physical state of lactose. Above this level, salts have a marked influence (Pisecky, 1997). The water activity of milk powders consisting of non-fat milk solids and milk fat is controlled mainly by the moisture content expressed in non-fat solids since the fat has no influence. Thus, differences in aw are due mostly to the state of proteins and the physical state of the lactose. The methods

Understanding the factors affecting spray-dried dairy powder

39

to determine aw consist of putting the product in equilibrium with the surrounding atmosphere, then measuring the thermohygrometric characteristics of the air in equilibrium with the product. The aw should be close to 0.2 at 25°C for optimal preservation (Efstathiou et al., 2002). Water activity plays an important role during the dehydration process, and an understanding of sorption isotherms can provide valuable guidelines for the engineering design, control of the drying process (isothermal desorption curves) and storage stability (isothermal adsorption curves). In practical terms, all dairy powder isotherms found in the literature were obtained using final products as starting materials. The powder or the concentrate is exposed to air of defined relative humidity and brought to equilibrium, after which the moisture content is determined. Thus, the published isotherms are designated as adsorption or desorption. Establishing equilibrium can very often take weeks. Many mathematical equations, both theoretical and empirical, have been reported in the literature to express the water sorption isotherms of dairy powders (Pisecky, 1997). With practical or theoretical sorption isotherms, the ideal moisture content can be determined for the optimal stabilization (at 0.2 aw and at 25°C) of some dairy powders. For example, the corresponding moisture content must be close to 4%, 2–3% and 6%, for skim milk, whey and caseinate powders, respectively. With Tg, water activity is one of the main factors governing many of the phenomena occurring during thermal dehydration, including: • Easiness of water evaporation from a liquid droplet, • Particle temperature history during the whole removal process, • Moisture content equilibrium that can be achieved under given conditions at infinite residence time, • Stickiness of the product (in relation to the Tg) and outlet conditions used for drying without occurrence of sticking (Pisecky, 1997).

2.4

Effects of spray drying parameters on powder properties

2.4.1 During spray drying The properties of the final product are influenced by a number of factors, including the quality and composition of the raw milk and the processing conditions applied. As some of the factors are subject to both seasonal and daily variations, it is necessary to monitor those properties that might be affected by these variations and to make appropriate corrections to the operation parameters. Two major properties, i.e. moisture content and bulk density, are good examples described below to show the strong relationships between process and products.

40

Dairy-derived ingredients Drying air parameters

Concentrate flow rate

Sprayer

AH of outlet air T°C of outlet air

Prior process

Nozzle / Wheel Heat treatment

Inlet air T°C and AH before and after heating

Concentrate residence time

RH

Concentrate temperature

T°C

H2O

aw Spray drying Agglomeration Stickiness–cakiness Rehydration

Ø Droplet

η of [C]

Homogenization Total solid content

Tg°C

Biochemical composition

Fig. 2.3 Influence of various factors on powder moisture content. AH = Absolute humidity; RH = Relative humidity; H2O = Moisture content; η = Viscosity; T°C = Temperature; Tg°C = Glass transition temperature; Ø = Size; [C] = Concentrate; aw = water activity

Moisture content Figure 2.3 shows the influence of various factors, such as the relative humidity (RH) of the outlet air, on powder moisture (Schuck et al., 2008; Pisecky, 1997). As shown in this Figure, moisture content depends on the drying air parameters, on the spray dryer and on the previous process. Moisture content is influenced by the aw, which depends on the RH of the outlet air. The relative humidity (RH) is influenced by the absolute humidity (AH) and temperature of the outlet air, which themselves depend on the concentrate flow rate, the inlet air temperature and AH before and after heating. The moisture content is also influenced by the size of the droplet, depending on the type of sprayer and viscosity. The viscosity of the concentrate depends on the heat treatment, temperature, residence time, homogenization, total solid content and biochemical composition. The temperature T of the droplet (and thus the powder) is influenced by the temperature and AH of the inlet air and by the RH, AH and temperature of the outlet air. The glass transition temperature (Tg) depends on biochemical composition, aw and moisture content. Knowledge of the T and Tg makes it possible to monitor agglomeration, stickiness, cakiness and rehydration. Experiments reported by Schuck et al. (2008) show that the outlet air temperature is not always the optimum parameter to affect the moisture content of a dairy powder. The RH of the outlet air is the key parameter to optimize the moisture content and water activity of dairy powders.

Understanding the factors affecting spray-dried dairy powder Feed composition

Feed concentration

Solids density Volume reduction Wheel

Spraying Feed temperature

41

Occluded air

Particle density

Air in droplets

Nozzle

Feed foaming ability

Feed aeration

Inlet temperature

Particle temperature history

Feed viscosity

Particle size distribution

Outlet temperature

Particle moisture

Flowability

Absolute humidity

Fines return

Bulk density

Interstitial air Milk preheating

Age thickening

Speed or pressure

Agglomeration

Fig. 2.4 Interrelationship of various drying parameters and physicochemical properties on bulk density (Pisecky, 1997; Carric´ and Milanovic´, 2003).

Bulk density Other important properties are bulk density and particle density. High bulk density is of economic importance in products for shipment in bulk, saving both packaging material and transport costs. Figure 2.4 shows the interrelationship of various drying parameters and physicochemical properties, and their relationship with bulk density (Pisecky, 1997; Carric´ and Milanovic´, 2003). Apart from the product mass density, which is provided by its composition, two factors have a decisive influence on bulk density, namely the particle density (provided by product mass density and the occluded air content), and the interstitial air content (provided by particle size distribution and agglomeration). Particle density depends on many factors. The composition of the solids plays an important part, first of all because it defines the product mass density. High protein content tends to reduce particle density as it increases the tendency of the feed to foam. This foaming can be suppressed somewhat by high heat pre-treatment, (denaturing the whey proteins), and also by high concentration combined with heating the feed. To achieve high particle density, it is important to avoid any treatment that may incorporate air into the feed, such as excessive agitation. Rotary atomizers tend to incorporate air into the droplets, and pressure nozzles produce much higher particle density than rotary wheels. However, special vane-shaped rotary wheels are now available with less tendency to entrap air in product droplets. The presence of air in the atomized droplets results in occluded air in the dried particles. Depending on drying conditions, or to be precise on the particle temperature during the drying process, any air bubbles initially present may expand and further reduce the particle density. Therefore, if

42

Dairy-derived ingredients 0.35

Tg Adsorption isotherm

50°C

50

0.30 0.25

20°C 0.20 0 0.15 8 g.100 g–1

–50

5 g.100

g–1

0.10

Water content (kg water kg–1 total solids)

Temperature (°C)

100

0.05

0.4 aw –100 0

Fig. 2.5

0.2

0.4 aw

0.6

0 0.8

Glass transition temperature (Tg) and water adsorption isotherm for skim milk solids and Tg curve of lactose (Roos, 2002).

all other conditions are the same, the two-stage or three-stage drying processes provide higher particle density than single-stage drying.

2.4.2 During storage From the thermodynamic information (Tg, aw, see Section 2.3.2), it should be possible to anticipate the behavior of a powder at a given temperature and under aw conditions. Combination of the Tg and aw curves in relation to the moisture content makes it possible to obtain a very interesting profile (Fig. 2.5) (Roos, 2002). From this curve, it is easy to determine the moisture content and the Tg at different aw values in relation to a skim milk powder. For example, the moisture content and the Tg at 0.2 aw, will be close to 5% and 50°C, respectively. If one wants an aw close to 0.4, the moisture content and the Tg will be close to 8% and 20°C, respectively. However, for this powder some enzymatic and non-enzymatic reactions can begin at 0.4 aw, which may increase deterioration and loss of quality. Moreover, it is not easy to stabilize this powder at a temperature lower than 20°C.

2.4.3 During rehydration As explained in Section 2.3.1, Rehydration, the IDF method, NMR spectroscopy, turbidimetry, viscosimetry and granulometry provide valuable information regarding the mechanisms involved in dairy powder dissolution with constant stirring. However, to optimize the rehydration time, two parameters are very important, namely particle size, which depends on the spray drying conditions, and the powder structure, in particular the surface composition.

Understanding the factors affecting spray-dried dairy powder

43

Effects of particle size As expected, granulation has a positive effect on wetting. The wetting time is systematically better for granulated particles. This phenomenon is well known, with large particles forming large pores, high porosity and small contact angles between the powder surface and the penetrating water as fast wetting is enhanced (Freudig et al., 1999). A surprising influence of granulation on the rehydration time was observed in the study of Gaiani et al. (2005). Depending on the nature of the protein, the granulation influence resulted in opposite effects. Whey protein isolate (WPI) rehydration time was shorter for granulated particles whereas it was shorter for non-granulated particles of micellar casein isolate (MCI). This was unexpected and could be explained by the controlling stage rate. The controlling stage for whey proteins is wetting. As granulation improves the wetting stage, the rehydration of whey powders is enhanced for granulated particles. In contrast, in this study, the controlling stage for casein proteins was dispersion. In fact, even with a shorter wetting time, a granulated powder is slower to rehydrate than a non-granulated powder (Gaiani et al., 2005). These results are not compatible with other studies on skim milk powder, in which it was generally accepted that a single particle size around 200 µm (Neff and Morris, 1968) or 400 µm (Freudig et al., 1999) represented optimal dispersibility and sinkability. In fact, this optimal particle size depends on the composition of the dairy powder. If the industry wishes to optimize powder rehydration, it seems to be better to rehydrate granulated powders when the protein is whey and to rehydrate non-granulated powders when the protein is casein. Effects of composition surface Gaiani et al. (2006b) have shown that X-ray photoelectron spectroscopy (XPS) analysis yields essential information complementary to standard methods, providing greater understanding of rehydration by determination of the surface composition. A small change in powder surface composition or bulk composition can totally change the rehydration properties. It is worth noting that powders with less distinct compositions might present significant rehydration differences. Table 2.1 shows the effects of storage on the surface composition of micellar casein powder and how lipids can migrate from the bulk to the surface. XPS has revealed that lipids are more likely to migrate toward the surface than lactose or proteins. We have demonstrated that the monolayer moisture capacity (Xm) determined by dynamic vapor sorption (DVS), which is decreased during storage, is probably due to the presence of surface lipids (formation of a hydrophobic surface) and that the presence of pores at the surface could be a route allowing lipid release. A fat layer could cover progressively the surface during storage, inducing a decrease of the monolayer capacity due to its hydrophobic nature.

44

Dairy-derived ingredients

Table 2.1 Monolayer moisture capacity (Xm), surface fat composition and wetting time modifications of micellar casein powders during storage at 20 and 50°C Storage temperature (°C) 20

50

Storage duration (days)

Xm (kg.kg−1)

Surface fat (%)

Wetting time (s)

0 15 30 60 0 15 30 60

0.0632 0.0626 0.0617 0.0617 0.0632 0.0574 0.0537 0.0524

6 6 12 16 6 6 13 17

12 14 66 73 12 15 68 265

The combination of these two methodologies (DVS and XPS) enhanced our understanding of lipid migration on a powder surface. This study also revealed the possibility of creating porosity inside the powder by controlled storage conditions (e.g. temperature, relative humidity, time). Further studies with powders containing more or less fat would therefore improve the understanding of fat release. In the future, XPS analysis could help to formulate dairy powders presenting better rehydration properties. Improving both the wetting time and the rehydration time could be one way to extend the applications of high protein content powders, the principal limitation of these powders being the low water transfer during rehydration (Gaiani et al., 2007).

2.5

Control and improvement of powder properties

Due to the variety and complexity of the mixes to be dried, a more rigorous method based on physico-chemical and thermodynamic properties has now become necessary. Improved understanding of the biochemical properties of milk products before drying, water transfer during spray drying, the properties of powders and influencing factors has now become indispensable in the production of dairy powders. Lack of technical and economic information and of scientific methods prevents the manufacturer from optimizing his process in terms of energy costs and powder quality. Two approaches are necessary for dairy research into the spray drying of dairy products, one involving the products (availability of water related to the biochemical composition) and the second involving the process (knowledge and improvement of the drying parameters). 2.5.1 Availability of water The aim of this section is to propose a new method (drying by desorption, using a thermohygrometer sensor) in order to determine major drying

Understanding the factors affecting spray-dried dairy powder

45

parameters according to food components in relation to their interactions with water (bound and free water) and linked to water transfer kinetics. Schuck and co-workers’ studies (1998, 2009) have shown that drying by desorption is an excellent tool to determine and optimize the major spraydrying parameters in relation to biochemical composition according to water availability and desorption behavior (calculation of extra energy ∆E). The experimental device proposed by the authors differs from spray-drying equipment in terms of duration of drying, drying temperature, surface/ volume ratio, etc. because the concentrate is dried in a cup and not in a droplet However, some computational tools have been developed to improve the method by taking these factors into account. Validation tests (>30 products) have indicated that this method could be applied to a wide range of food products and spray-dryer types. For reasons of calculation speed and reliability, this method has been computerized and it can already be used in the determination of parameters of spray drying for food products. The name of the new software is ‘Spray Drying Parameter Simulation and Determination Software’ (SD2P®), registered under the following identification: IDDN.FR.001.480002.002.R.P.2005.000.30100. Analysis of the desorption curve (measured relative humidity versus time), combined with knowledge of the temperature, total solids, density and specific heat capacity of the concentrate, air flow rates, theoretical water content in relation to water activity and RH of the outlet air, current weather conditions, cost per kWh and percentage of drying in the integrated fluid allows determination of enthalpy, T, RH (including ∆E) for each inlet air, concentrate and powder flow rate, specific energy consumption, energy and mass balance, yield of the dryer and cost (in * or in $) to remove 1 kg of water or to produce 1 kg of powder. All these results are summarized in Fig. 2.6. This figure is a representation of the software delivery: • air characteristics at the dryer/integrated fluid bed inlet and outlet (upper part), • flow, energy and cost calculations (lower part) (Schuck et al., 2009)

2.5.2 Process improvement The aim of this section is to show the use of a thermohygrometric sensor, with some examples of such measurements (temperature, absolute (AH) and relative humidity (RH), dry air flow rate, water activity) through calculation of mass and absolute humidity to prevent sticking in the drying chamber and to optimize powder moisture and water activity in relation to the relative humidity of the outlet air. It was demonstrated by Schuck et al. (2005b) that a thermohygrometer can be used to avoid sticking and to optimize water content and water activity in dairy powders. From these results, it could be seen that the

46

Dairy-derived ingredients

Skim Milk Concentrate Mass flow rate (kg DA/h)

Reduce Inlet air before heating

Enthalpy (kJ/kg DA)

Temperature (°C)

AH (g/kg DA)

RH (%)

32.8

20

5

34.2

Inlet air after heating ‘I’

80000

259.5

242.3

5

0.02

Cooling air ‘C’

3000

32.8

20

5

34.2

Recirculation air ‘R’

3000

32.8

20

5

34.2

Complementary air ‘C’

1000

32.8

20

5

34.2

Air mix (I+C+R)

87000

241.3

223.5

5

0.03

Outlet air 1 stage (I+C+R+C)

87000

212.3

81

49.1

15

Integrated fluid bed inlet air ‘B’

10000

107.2

92.8

5

1

Integrated fluid bed outlet air ‘B’

10000

81.4

49.2

12.2

16.3

Overall outlet air (I+C+R+C+B)

97000

198.8

77.9

45.3

15.7

Evaporation capacity (kg/h)

3908.7

Water flow rate in concentrate (kg/h)

4065

Wet bulb temperature of overall outlet air (°C)

47.9

Dew temperature of overall outlet air (°C)

38.9

Concentrate flow rate (kg/h)

8468.8

Energy balance (kJ/kg water)

4646

Concentrate flow rate (l/h)

6941.7

Energy consumption ratio (60°C) (kg vapour/kg water) Yield (60°C) (%)

50.8

Concentrate density (-)

1.22

2

Concentrate dry matter (%)

52

Cost ($/ton water)

77.4

Powder moisture (%)

4

Cost ($/ton powder)

68.6

kWh cost ($)

0.06

Powder flow rate (kg/h)

4587.3

Concentrate temperature (°C)

45

Concentrate Cp (kJ/(kg°C))

3.5

Fig. 2.6

Default

Print

Export

Quit

Parameters of spray drying calculated by the SD2P® software.

calculated AH is systematically higher than the measured AH, because the calculated AH corresponded to the maximum theoretical value that could be reached. Calculation of AH by means of the mass balance is based on the hypothesis that the air circulating in the spray drier removes all the water from the concentrate. Thus, if the difference between calculated and measured absolute humidity of the outlet air is below 2 g of water per kg dry air (depending on the spray drier with regard to measurement accuracy), there is no problem of sticking in the spray dryer chambers, whatever the dairy concentrate used. On the other hand, sticking was observed in this study for differential AH above 2 g water per kg dry air, corresponding to lower water removal and consequently to favorable sticking conditions. The operator can follow the absolute humidity and anticipate a variation in drying parameters according to the differences between calculated and measured absolute humidity.

Understanding the factors affecting spray-dried dairy powder

47

The operator can also follow the relative humidity in the outlet air. To achieve a dairy powder with the same water activity and moisture content, he must always maintain the same relative humidity in the outlet air according to each dairy product, whatever the spray drying conditions (inlet air temperature, relative and absolute humidity). The changes in relative and absolute humidity (resulting from variations in absolute humidity of inlet air, total solid content of concentrate, crystallization rate, outlet air temperature, etc.) can be rapidly observed in the outlet air using a thermohygrometer before such changes significantly affect powder moisture, water activity and powder behavior with regard to sticking.

2.6

Conclusions and future trends

This chapter describes the effects of spray drying on the quality of dairy powders during drying and rehydration. It also demonstrates that the quality of these powders depends on the interactions between the biochemical composition and the process parameters. Fig. 2.7 shows that the biochemical, microbiological and physical properties of a dairy powder and its recombined product depend on many parameters. For example: Crystallization Concentration Homogenization Pre-treatment

Processes

Spray drying and fluidization Production

Physical properties

Rehydration Powder

Biochemical properties Microbiological qualities

Rehydration conditions

Storage conditions

Composition and/or water availabilty

Stability

Fig. 2.7

Properties of dairy powders in relation to production, storage and rehydration.

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Dairy-derived ingredients

• the pre-heat treatment process parameters, concentration by membrane filtration or by vacuum evaporation, crystallization, homogenization, spray drying and fluidization, • the storage conditions (relative humidity, temperature, packaging, . . .) to optimize stability over to time, • the rehydration conditions (stirring conditions, temperature, concentration, . . .) to improve water transfer to obtain the best quality recombined product from the corresponding powder. Moreover, the biochemical composition (nature and content) and water availability interact in all the stages of production, stability and rehydration. The quality of a dairy powder can therefore be improved only if research is undertaken on the process–product interactions. In conclusion, this chapter discusses the complexity of the spray drying process for dairy manufacturers who want to optimize their production. It is impossible to provide a simple mathematical model that takes into account all parameters simultaneously for the complex processes in the spray drying of dairy products. Furthermore, existing studies, essentially centered on skim milk, cannot easily be extrapolated to other dairy products. Study of more interaction processes, structures and functions of dairy products is necessary in order to increase our understanding of the mechanisms of water transfer, drying parameters, storage conditions and rehydration of dairy powders with thermodynamical and biochemical approaches.

2.7

References

aguilar ca and ziegler gr (1993), ‘Lactose crystallization in spray-dried milk powders exposed to isobutanol’, Food Struct, 12, 43–50. american dairy products institute (1990), Standards for Grades of Dry Milk Including Methods of Analysis, Chicago, American Dairy Products Institute. baldwin aj, baucke ag and sanderson wb (1980), ‘The effect of concentrate viscosity on the properties of spray dried skim milk powder’, N Z J Dairy Sci Technol, 15, 289–297. bimbenet jj, schuck p, roignant m, brulé g and méjean s (2002), ‘Heat balance of a multistage spray-dryer: Principles and example of application’, Lait, 82, 541–551. boonyai p, bhandari b and howes t (2004), ‘Stickiness measurement techniques for food powders: A review’, Powder Technol, 145, 34–46. boonyai p, bhandari b and howes t (2005), ‘Measurement of glass–rubber transition temperature of skim milk powder by static mechanical test’, Drying Technol, 23, 1499–1514. caric´ m (2003), ‘Types and Manufacture’, in Roginsky H, Encyclopedia of Dairy Sciences, London, Academic Press, 1869–1874. caric´ m and kaláb m (1987), ‘Effects of drying techniques on milk powders quality and microstructure: A review’, Food Microstruct, 6, 171–180. caric´ m and milanovic´ (2003), ‘Physical and functional properties of milk powders’, in Roginsky H, Encyclopedia of Dairy Sciences, London, Academic Press, 1874–1880.

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49

carr rl jr (1965), ‘Evaluating flow properties of solids’, Chem Eng, 72, 163–168. couchman pr and karasz fe (1978), ‘A classical thermodynamic discussion of the effect of composition on glass transition temperatures’, Macromol 11, 117–119. davenel a, schuck p and marchal p (1997), ‘A NMR relaxometry method for determining the reconstitutability and the water-holding capacity of protein-rich milk powders’, Milchwissenschaft, 52, 35–39. de vilder j (1986), ‘La fabrication de poudre de lait écrémé instantanée. I. Les caractéristiques physiques et chimiques’, Rev Agric, 39, 865–877. efstathiou t, feuardent c, méjean s and schuck p (2002), ‘The use of carbonyl analysis to follow the main reactions involved in the process of deterioration of dehydrated dairy products: Prediction of most favourable degree of dehydration’, Lait, 82, 423–439. fauquant j, maubois jl and pierre a (1988), ‘Microfiltration du lait sur membrane minérale’, Tech Lait, 1028, 21–23. freudig b, hogekamp s and schubert h (1999), ‘Dispersion of powders in liquid in a stirred vessel’ Chem Eng and Process, 38, 525–532. gaiani c, banon s, scher j, schuck p and hardy j (2005), ‘Use of a turbidity sensor to characterize casein powders rehydration: Influence of some technological effects’, J Dairy Sci, 88, 2700–2706. gaiani c, scher j, schuck p, hardy j, desobry s and banon s (2006a), ‘The dissolution behaviour of native phosphocaseinate as a function of concentration and temperature using a rheological approach’, Int Dairy J, 16, 1427–1434. gaiani c, ehrhardt jj, scher j, hardy j, desobry s and banon s (2006b), ‘Surface composition of dairy powders observed by X-ray photoelectronspectroscopy and effects on their rehydration properties’, Coll Surf B Biointer, 49, 71–78. gaiani c, schuck p, scher j, hardy j, desobry s and banon s (2007), ‘Dairy powder rehydration: Influence of proteins and some technological effects’, J Dairy Sci, 90, 570–581. hardy j, scher j and banon s (2002), ‘Water activity and hydration of dairy powders’, Lait, 82, 441–452. haugaard sorensen i, krag j, pisecky j and westergaard v (1978), Méthodes d’analyses desproduits laitiers déshydratés, Copenhagen, Niro A/S. international dairy federation (1988), Dried Milk and Milk Products – Determination of Insolubility Index. IDF Standard 129A, Brussels, International Dairy Federation. jeantet r, schuck p, famelart mh and maubois jl (1996), ‘Intérêt de la nanofiltration dans la production de poudres de lactosérum déminéralisées’, Lait, 76, 283–301. jeantet r, croguennec t, mahaut m, schuck p and brulé g (2008a), Les Produits Laitiers, Paris, Technique et Documentation – Lavoisier, jeantet r, ducept f, dolivet a, méjean s and schuck p (2008b), ‘Residence time distribution: A tool to improve spray-drying control’, Dairy Sci Technol, 88, 31–43. jouppila k and roos yh (1994), ‘Water sorption and time-dependent phenomena of milk powders’, J. Dairy Sci. 77, 1798–1808. king n (1966), ‘Dispersibility and reconstitutability of dried milk’, Dairy Sci Abs 28, 105–118. masters k (1991), Spray drying, Essex, Longman Scientific & Technical. masters k (2002), Spray drying in practice, Charlottenlund SprayDryConsult International ApS. maubois jl (1991), ‘New applications of membrane technology in the dairy industry’, Aust J Dairy Technol 46, 91–95.

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mistry vv, hassan hn and robinson dj (1992), ‘Effect of lactose and protein on the microsctructure of dried milk’, Food Struct 11, 73–82. neff e and morris ha (1968), ‘Agglomeration of milk powder and its influence on reconstitution properties’, J Dairy Sci, 51, 330–338. pierre a, fauquant j, le graët y, piot m and maubois jl (1992), ‘Préparation de phosphocaséinate natif par microfiltration sur membrane’ Lait, 72, 461–474. pisecky j (1980), ‘Bulk density of milk powders.’, Aust J Dairy Technol, 35, 106–111. pisecky j (1981), ‘Technology of skimmed milk drying’, J Soc Dairy Technol, 34, 57–62. pisecky j (1986), ‘Standards, specifications and test methods for dry milk products’, In MacCarthy D, Concentration and Drying of Food, London, Elsevier, 203–220. pisecky j (1990), ‘20 years of instant whole milk powder’, Scand Dairy Inf, 4, 74–77. pisecky j (1997), Handbook of Milk Powder Manufacture, Copenhagen, Niro A/S. refstrup e (2003), ‘Drying of Milk’, in Roginsky H, Encyclopedia of Dairy Sciences, London, Academic Press, 860–871. roos yh (2002), ‘Importance of glass transition and water activity to spray drying and stability of dairy powders’, Lait, 82, 478–484. schuck p, piot m, méjean s, fauquant j, brulé g and maubois jl (1994a), ‘Déshydratation des laits enrichis en caséine micellaire par microfiltration; Comparaison des propriétés des poudres obtenues avec celles d’une poudre de lait ultrapropre’, Lait, 74, 47–63. schuck p, piot m, méjean s, le graët y, fauquant j, brulé g and maubois jl (1994b), ‘Déshydratation par atomisation de phosphocaséinate natif obtenu par microfiltration sur membrane’, Lait, 74, 375–388. schuck p, roignant m, brulé g, davenel a, famelart mh and maubois jl (1998), ‘Simulation of water transfer in spray drying’, Drying Technol, 16, 1371–1393. schuck p, blanchard e, dolivet a, méjean s, onillon e and jeantet r (2005a), ‘Water activity and glass transition in dairy ingredients’, Lait, 85, 295–304. schuck p, méjean s, dolivet a and jeantet r (2005b), ‘Thermohygrometric sensor: A tool for optimizing the spray drying process’, Innov. Food Sci Emerg Technol, 6, 45–50. schuck p, méjean s, dolivet a, jeantet r and bhandari b (2007), ‘Keeping quality of dairy ingredients’, Lait, 87, 481–488. schuck p, dolivet a, méjean s and jeantet r (2008), ‘Relative humidity of outlet air: Key parameter to optimize moisture content and water activity of dairy powder’, Dairy Sci Technol, 88, 45–52. schuck p, dolivet a, méjean s, zhu p, blanchard e and jeantet r (2009), ‘Drying by desorption: A tool to determine spray drying parameters’, J Food Eng, 94, 199–204. sougnez m (1983), ‘L’évolution du séchage par atomisation’, Chim Mag, 1, 1–4. tuohy jj (1989), ‘Some physical properties of milk powders’ Irish J Food Sci Technol, 13, 141–152. westergaard v (2003), ‘Dryer design’, in Roginsky H, Encyclopedia of Dairy Sciences, London, Academic Press, 871–889. westergaard v (2004), Milk powder technology – Evaporation and spray-drying, Copenhagen, A/S Niro A/S.

3 Production and enrichment of bioactive peptides derived from milk proteins L. Vercruysse, J. Van Camp, K. Dewettinck and G. Smagghe, Ghent University, Belgium

Abstract: In this chapter, information is given on the production of bioactive peptides from milk proteins. After defining bioactive peptides, the necessity to release bioactive peptides from the parent protein is discussed. Then, the two main approaches for release, namely enzymatic hydrolysis and microbial fermentation are reviewed. Next, an overview is given of the main unit operations that can be applied for the enrichment and selective isolation of bioactive peptides from the protein hydrolysates, including membrane filtration, chromatography and precipitation. Attention is given mainly to ACE-inhibitory peptides, and to processes that show potential for application in the dairy industry. Key words: milk, bioactive peptides, enzymatic hydrolysis, microbial fermentation, enrichment.

3.1

Introduction

3.1.1 Defining bioactive peptides Food proteins are traditionally considered as sources of both energy and of building blocks for growth and maintenance of the body. However, recent data indicate that food proteins are also unique sources of bioactive sequences (Murray and FitzGerald, 2007). Biologically active peptides, also called bioactive peptides, exert physiological functions in the body resulting in beneficial effects towards health (Kitts and Weiler, 2003). Many bioactive peptides are latent in that they are inactive in the parent protein sequence and need to be released to exert their activity. There are two main approaches for releasing bioactive peptides. One approach is to use lactic acid bacteria (LAB) for fermentation. The other approach is to subject food proteins to in vitro hydrolysis by one or a combination of enzymes (Lopez-Fandino et al., 2006). Bioactive peptides are short chains of amino

52

Dairy-derived ingredients

acids, usually containing two to 20 amino acid residues per molecule. A wide range of activities has been described, including blood pressure lowering (angiotensin converting enzyme (ACE) inhibitory), opioid, antimicrobial, mineral binding, antioxidant, antithrombotic, immunomodulatory and anticancer effects. Many peptides are known to reveal multifunctional properties as they can exert more than one biological effect (Hartmann and Meisel, 2007).

3.1.2 Bioactive peptides in milk Numerous food proteins of both animal and vegetable origin have been used to generate bioactive peptides. Plant protein sources of bioactive peptides include maize, soybean, wheat and rice (Yamamoto et al., 2003; Murray and FitzGerald, 2007). Animal proteins, such as bovine, chicken and porcine muscle, contain bioactive peptides as well as fish proteins from bonito, sardine, tuna and salmon (Vercruysse et al., 2005). However, the major source of bioactive peptides identified to date is milk. The most important is bovine milk, but also human, sheep (Papadimitriou et al., 2007), goat, pig, buffalo and yak milk (Mao et al., 2007) have been reported as sources of bioactive peptides (Minervini et al., 2003). The main constituents of milk are casein and whey proteins. Bovine casein accounts for about 80% of the total milk proteins and is further divided into α-, β- and κ-casein. The major bioactive peptides derived from casein, exerting a role in the nervous, cardiovascular, digestive and immune system, have been reviewed by Silva and Malcata (2005). In addition, whey, which is a major product of the modern cheese and casein industry, is an important source of bioactive peptides exerting various activities (Smithers, 2008). The major whey proteins and peptides, such as β-lactoglobulin (β-Lg), α-lactalbumin and glycomacropeptide are known to positively influence human health (Chatterton et al., 2006; Hernandez-Ledesma et al., 2008). Milk, fermented milk, yoghurt and cheese, but also infant milk formulas (De Noni, 2008; Martin et al., 2008) have been identified as sources of bioactive peptides. Milk proteins are attractive in the search for bioactive peptides as they are very well characterized and are available in large amounts at a high degree of purity and at a low price.

3.1.3 Necessity to release bioactive peptides from milk proteins In past years, the protein fraction in milk has received great attention and several protein components exerting potential health-promoting activity have been identified and characterized. Intact milk proteins have been associated with a wide range of activities, including iron binding (e.g. lactoferrin) and antimicrobial effects (e.g. lactoperoxydase, lysozyme) (Haque and Chand, 2008). However, much of the biological activity of milk proteins is latent. The bioactive peptides have to be released from precursor milk

Production and enrichment of bioactive peptides

53

proteins in one of the following ways: (a) enzymatic hydrolysis by digestive enzymes, (b) fermentation with proteolytic starter cultures, (c) proteolysis by enzymes derived from microorganisms or plants and (d) proteolysis by indigenous proteases present in milk (Korhonen and Pihlanto, 2007). Synergistic effects between intact bioactive milk proteins and bioactive milk-derived peptides are important as both compounds might be simultaneously present in the human gastrointestinal tract. Lopez-Exposito et al. (2008) indicated such a synergistic effect between lactoferrin and the antimicrobial peptide lactoferricin against Escherichia coli and Staphylococcus epidermidis.

3.2

Enzymatic hydrolysis

Enzymatic hydrolysis is the most common way to release bioactive peptides from milk proteins. One or a combination of enzymes can be used in a batch or continuous process. In vitro hydrolysis of milk proteins allows the selection of the protein substrate and enzyme specificity to optimize the yield of bioactive peptides. Highly effective in the enzymatic release of bioactive peptides from milk proteins are pancreatic proteinases, especially trypsin. Other endoproteinases such as chymotrypsin, pepsin and thermolysin, and proteinase extracts derived from bacteria, moulds, plants and animals have also been used (Korhonen and Pihlanto, 2007). In comparison to digestive enzymes, microbial enzymes use different cleavage sites; thus, peptides liberated by these enzymes may differ from those liberated by digestive enzymes. Mizuno et al. (2004) found in a comparative study of nine commercial enzymes that an Aspergillus oryzae protease was most effective for the release of short and proline-rich anti-hypertensive peptides from casein. However, caseinates derived from milk of different species (bovine, sheep, goat, pig, buffalo, human) can show differences in susceptibility towards enzymatic hydrolysis, as demonstrated by Minervini et al. (2003) who used a partially purified serine proteinase from Lactobacillus helveticus. This result can be explained by differences in sequence identity, conformation, and relative proportions of individual caseins. Different species were shown to generate different types of peptides with ACE-inhibitory and antimicrobial activity (Minervini et al., 2003). A combination of digestive enzymes will alter the digestive process and the peptides released, but will not necessarily result in a higher activity. Mullally et al. (1997) used gastric and pancreatic proteases to hydrolyze whey proteins in order to obtain ACE inhibitory peptides. Hydrolysis of α-lactalbumin and β-Lg with pepsin and trypsin separately resulted in similar ACE inhibitory activities. Similar activities were also obtained when using combinations of trypsin and pepsin; moreover, the order of addition of trypsin and pepsin had little effect on the ACE inhibitory values. However, the types of peptides present in the

54

Dairy-derived ingredients

final hydrolysate may be affected by the order of enzyme addition (Mullally et al., 1997). Gastro-intestinal digestion can be a way to release bioactive peptides from milk proteins. However, strong hydrolysis during gastrointestinal digestion and absorption can limit the bioavailability of target peptides, which makes their applicability for systemic functions more difficult (Vermeirssen et al., 2004a). The gastro-intestinal digestion process can be simulated in batch or semi-continuous reactor systems (Vermeirssen et al., 2003). The production of bioactive peptides using enzymatic hydrolysis can be studied using response surface methodology. Van der Ven et al. (2002) showed that the ACE inhibitory activity of whey protein hydrolysates could be controlled by systematical regulation of process conditions. Roufik et al. (2006) demonstrated that the in vitro digestibility of bioactive peptides derived from bovine β-Lg is not only influenced by pH, time of incubation, and enzyme/substrate ratio, but also by the length and the nature of the peptides. Also, other peptides present in the medium can influence the hydrolysis by gastro-intestinal enzymes, e.g. a higher degree of hydrolysis (DH) was obtained for pepsin and chymotrypsin when the opioid agonistic and ACE inhibitory peptide β-Lg f102-105 was combined with β-Lg f92105, than when both peptides were hydrolyzed separately. It was suggested that in a mixture, peptides may form complexes that interact more efficiently with the hydrolyzing enzymes, compared with peptides alone (Roufik et al., 2006). El-Zahar et al. (2005) also demonstrated that the enzymatic susceptibility towards pepsin of ovine β-Lg was significantly higher compared with that of bovine β-Lg. The effect was attributed to a slightly different tertiary structure and a higher surface hydrophobicity at pH 2 for ovine β-Lg. Especially, the more hydrophobic C-terminal end of ovine β-Lg may be more exposed at the surface of the molecule, making it susceptible to pepsin hydrolysis. A more industrial approach to the production of bioactive peptides is enzymatic digestion in membrane reactors. Bouhallab et al. (1993) designed a continuous process in which enzymatic hydrolysis of casein was combined with purification of the bioactive peptides through the use of ultrafiltration. In addition, the immobilization of proteases can lead to a better hydrolysis efficiency. Different proteases, such as trypsin, chymotrypsin, papain, and cathepsins have been successfully immobilized on different supports. Immobilization has several advantages, such as capability of reuse, better control of hydrolysis rate and longer stability of the enzyme (Toldra et al., 2005). The amino acid sequence of milk proteins has been reported, and can be used to evaluate the presence of bioactive peptides by computer-aided models. In silico screening was used by Vermeirssen et al. (2004b) to rank individual milk proteins according to their potential to generate ACEinhibitory peptides. A database containing the amino acid sequence of milk proteins was combined with a database containing the sequence and IC50-

Production and enrichment of bioactive peptides

55

values of 498 ACE-inhibitory peptides reported in literature. Proteins were given weighed scores on the basis of high ACE-inhibitory activity (Score 1), as well as on short peptide size combined with high ACE-inhibitory activity (Score 2). Fifty different ACE-inhibitory peptides were identified in β-Lg, giving it the highest ranking among the milk proteins both by Scores 1 and 2. Other milk proteins with high scores were lactotransferrin and β-casein. A similar technique was developed by Dziuba et al. (2004). Again, a database containing protein and bioactive peptide sequences enables searching for protein fragments identical to bioactive peptides among protein amino acid sequences. Theoretical predictions and simulations are considered as an emerging tool in peptide science. Although these models give some insight into the number and type of active peptide sequences present in milk and other food proteins, because they do not take into account the release of active peptides from the source protein, their results have to be interpreted with caution (Minkiewicz et al., 2008).

3.3

Microbial fermentation

Many industrially used dairy starter cultures are highly proteolytic. The peptides and amino acids degraded from milk proteins during fermentation contribute to the typical flavour, aroma and texture of dairy products. The proteolytic system of LAB can contribute to the liberation of bioactive peptides during fermentation. The cell-wall bound proteinases, which have a very broad substrate specificity, initiate the proteolytic attack and release of oligopeptides. In addition, transport systems specific for amino acids and peptides up to 18 amino acids are present in the LAB. Oligopeptides that cannot be transported into the cells can be further degraded after lysis of the bacterial cell, which allows the release of intracellular peptidases, such as di-, tri-, amino- or endopeptidases (Lopez-Fandino et al., 2006). It is important to choose the right strains or combination of strains with optimal proteolytic activity and lysis at the right time, as the number of bioactive peptides relies on a balance between their formation and breakdown into inactive peptides and amino acids (Gobbetti et al., 2000). In addition to a high number of LAB, some yeasts have also been used to ferment milk proteins in order to release bioactive peptides (see Table 3.1). Fermented milk, and peptides isolated from fermented milk, have been demonstrated to have various biological activities. LeBlanc et al. (2002) detected immunostimulatory peptides in milk fermented with a L. helveticus strain. An antioxidative peptide, determined as Ala-Arg-His-Pro-His-ProHis-Leu-Ser-Phe-Met, was isolated from κ-casein after fermentation with Lactobacillus delbrueckii subsp. bulgaricus IFO13953. Although the scavenging activity of the DPPH radical of the peptide was lower than butylated hydroxytoluene (BHT), the peptide showed about five times stronger antioxidative activity than BHT in the beta-carotene decolorization system

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Dairy-derived ingredients

Table 3.1 peptides

Examples of micro-organisms used for the production of bioactive

Micro-organism

Bioactivity

Representative references

Lactobacillus helveticus

ACE inhibition

Nakamura et al., 1995a; LeBlanc et al., 2002; Minervini et al., 2003; Jauhiainen et al., 2005 Singh and Chand, 2006 LeBlanc et al., 2002 Ong and Shah, 2008 Gomez-Ruiz et al., 2002 Ryhanen et al., 2001 Hayes et al., 2007 Algaron et al., 2004

Lactobacillus casei Lactobacillus plantarum Lactobacillus acidophilus Lactobacillus animalis Lactococcus lactis subsp. lactis Lactococcus lactis subsp. cremoris Lactobacillus delbrueckii subsp. bulgaricus Streptococcus thermophilus Saccharomyces cerevisiae Klyveromyces marxianus var. marxianus Enterococcus faecalis Leuconostoc mesenteroides subsp. dextranicum

Antioxidant Immunomodulatory ACE inhibition ACE inhibition ACE inhibition ACE inhibition Immunomodulatory and antihypertensive ACE inhibition Antioxidative ACE inhibition ACE inhibition ACE inhibition ACE inhibition ACE inhibition ACE inhibition

Gobbetti et al., 2000 Ashar and Chand, 2004; Chobert et al., 2005 Gobbetti et al., 2000 Chobert et al., 2005; Tsai et al., 2008 Nakamura et al., 1995a Belem et al., 1999 Miguel et al., 2006; u Qiros et al., 2007 Gomez-Ruiz et al., 2002

(Kudoh et al., 2001). Virtanen et al. (2007) investigated the production of antioxidant activity during fermentation of whey with 25 LAB strains. They indicated that the development of antioxidant activity was strain-specific with Leuconostoc mesenteroides ssp. cremoris strains, Lactobacillus jensenii (ATCC 25258) and Lactobacillus acidophilus (ATCC 4356) showing the highest activity. In addition, ACE inhibitory peptides released by fermentation of milk proteins have been reported in many studies and have been reviewed by Haque and Chand (2008). Nakamura et al. (1995a,b) identified two ACE inhibitory peptides, Val-Pro-Pro and Ile-Pro-Pro, derived from milk by fermentation with a combination of L. helveticus CP790 and Saccharomyces cerevisiae. These two peptides, of casein origin, exert in vivo antihypertensive activity in spontaneously hypertensive rats (SHR) and in hypertensive subjects (Takano, 1998). A fermented milk product containing these peptides was marketed by the Japanese Calpis company under the

Production and enrichment of bioactive peptides

57

trade name Amiiru S. A European fermented milk drink with blood pressure lowering activity, Evolus from Valio Ltd, Finland, is produced by fermentation with another strain, namely L. helveticus LBK 16H but contains the same two active peptides (Tuomilehto et al., 2004). PihlantoLeppala et al. (1998) studied the release of in vitro ACE inhibitory activity of whey and casein by fermentation with commercial yoghurt starters. They indicated that fermentation alone was not enough to generate ACE inhibitory activity, but a further digestion with pepsin and trypsin was necessary. A number of studies has shown that during cheese making, a wide range of peptides are released and many of them have exhibited biological activities, such as ACE inhibitory activity. The origin of the milk and the manufacturing conditions, as well as the cheese-ripening time, determine the release of peptides and thus the activity (Korhonen and Pihlanto, 2003). In middle-aged Gouda cheese, a higher ACE inhibitory activity was detected compared to short-termed or long-termed ripened cheese. In ripened cheese, the ACE inhibitory activity probably results from a complex mixture of ACE inhibitory peptides. The activity increases during cheese maturation, but decreases when the proteolysis exceeds a certain level (Meisel et al., 1997). Similarly, the free peptides from eight months-aged Gouda cheese exerted the strongest blood pressure lowering effect after oral administration in SHR, compared to 24 months-aged Gouda, Emmental, Blue, Camembert, Edam and Havarti cheese (Saito et al., 2000). Ong and Shah (2008) studied the effect of probiotic adjuncts on ACE inhibitory activity of Cheddar cheeses. Cheeses made with the addition of Lactobacillus casei 279, L. casei LAFTI®L26 or L. acidophilus LAFTI®L10 had significantly higher ACE inhibitory activity than those without any probiotic adjunct. The probiotic adjuncts were added to improve proteolysis and enhance flavour during Cheddar cheese ripening. The two ACE inhibitory peptides Val-Pro-Pro and Ile-Pro-Pro have also been detected in various Swiss cheeses. The milk pretreatment, cultures, scalding conditions and ripening time were again identified as the key factors influencing the concentration of these two peptides in cheese. On average, the cheese varieties contained similar concentrations of the peptides as the commercial fermented milk products (Butikofer et al., 2008). Certain cell envelope-associated proteinases (CEP) of LAB can release bioactive health-beneficial peptides during milk fermentation. Hebert et al. (2008) characterized the peptide sequence in the hydrolysis pattern of αand β-casein made by a CEP of L. delbrueckii subsp. lactis CRL 581. In this pattern, a series of potentially bioactive peptides could be detected. The antihypertensive tripeptide Ile-Pro-Pro can be released by L. delbrueckii subsp. lactis CRL 581; therefore, the micro-organism could be used as a functional cheese starter. A combination of lactic acid fermentation and proteolysis of milk can be used to obtain a product which is more abundant in bioactive peptides

58

Dairy-derived ingredients

than the traditional fermentation or proteolysis products of milk. Tsai et al. (2008) fermented milk using two LAB (Streptococcus thermophilus and Lactobacillus bulgaricus) and added flavourzyme at the beginning of the fermentation. This combination accelerated the production of peptides (32.8 mg·g −1 peptides for the combination in contrast to 5.8 mg·g −1 peptides for the lactic fermentation alone) and increased the ACE inhibitory activity (IC50 value of 0.266 mg·mL −1 for the combination compared to 0.515 mg·mL −1 for the lactic fermentation alone). Similarly, Chen et al. (2007) fermented milk with LAB and added a protease, prozyme 6, five hours after the beginning of fermentation. The prozyme-facilitated fermentation resulted in a higher ACE inhibitory activity compared to the non-facilitated fermentation (Chen et al., 2007).

3.4

Enrichment of bioactive peptides

Various technologies have been applied for the enrichment and separation of bioactive peptides from the hydrolysates of milk proteins. However, commercial production has been limited by a lack of suitable large-scale technologies. Membrane filtration shows most potential to be used in pilot and industrial scale separations. Precipitation and chromatography are also available but are less frequently applied, mainly due to their lower selectivity and/or higher cost. The next sections will highlight a few recent evolutions in the use of membrane processing and chromatography for the separation and isolation of milk protein-derived bioactive peptides.

3.4.1 Membrane processing Peptides can be separated from protein hydrolysates using suitable membrane technologies, such as nano- and ultrafiltration. In membrane reactors, hydrolysis of milk proteins is combined with enrichment of the bioactive peptides from the reaction mixture through the use of filtration. Ultrafiltration membrane reactors yield a uniform product with desired molecular mass. Stepwise filtration has been found useful to separate small peptides. Pihlanto-Leppala et al. (2000) fractionated ACE-inhibitory peptides from α-lactalbumin and β-Lg hydrolysates using membranes with molecular weight cut-off (MWCO) of 1 and 30 kDa. IC50-values for ACE-inhibitory activities ranged from 109–837 µg·mL −1 in the 95%) although traces of lanosterol, dihydrolanosterol and β-sitosterol are also present (Jensen, 1973). 4.2.3 Factors influencing the milk lipids composition The chemical composition of milk fat is influenced primarily by feeding, the breed of the cow (genetics), its stage of lactation (physiological condition) and possible infections of the udder (mastitis). Actually, when all influencing factors are kept constant, differences in milk composition of different individuals can still be noticed (Walstra et al., 2006).

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Table 4.3 Chemical and physical properties of winter- and summer-fat (Bornaz et al., 1993; Hettinga, 1996; Vanhoutte, 2002) Fatty acids Samples

Average of total Summer Winter

Polyunsaturated

Iodine number

Volatile

Saturated

Monounsaturated

10.98

56.50

29.81

2.50

32.2

9.49 12.45

58.82 59.15

33.53 26.15

3.14 1.86

36.8 27.7

Firmness (N)

1.5–3 7–9

The most important factor of variability is feeding. An excellent illustration of this is the variation between summer and winter milk fat (Vanhoutte, 2002). Table 4.3 illustrates that when cows change from stall feeding (silaged feed) in the winter to fresh grass during the summer months, a raise in iodine value, a change in fatty acid composition and a decrease of hardness can be detected. Moreover, adding trisaturated triglycerides to the diet alters the fatty acid composition of the milk fat. Due to the occurrence of biohydrogenation in the rumen, the relation between unsaturated fatty acids in feed and their concentration in the milk fat is less obvious (Grummer, 1991; Vanhoutte, 2002). The physical form of feed also has an effect. If unsaturated fatty acids should be present in a specific matrix that can escape biohydrogenation, it can result in a higher proportion of unsaturated fatty acids in milk fat (Walstra et al., 2006). Moreover, several minor components, such as carotene and fat-soluble vitamins, are strongly affected by their content in the feed. The same counts for some trace elements. The breed of the cow is mainly selected by man, according to the intended use and the local conditions, such as climate, feed, terrain and customs, resulting in some variability in milk composition. Concerning the lactation period, the proportion of short chains is, for example, low at the start and increases until at least the first 8 to 10 weeks of the lactation period (Palmquist et al., 1993).

4.3

Milk fat modification

4.3.1 Introduction Anhydrous milk fat (AMF) was produced as an excellent transport and storage form of the large stocks of butter during the 1980s in the European Union and the United States. By removing the water phase from butter, milk fat could be stored for a considerably longer period of time without significant loss of quality. Nowadays, milk fat has acquired a certain market

Processing means for milk fat fractionation and production

73

share as a raw material for the confectionery and bakery industries, due to its unique organoleptic properties and its image as a natural ingredient (Kaylegian and Lindsay, 1995; Vanhoutte et al., 2002a). However, AMF has disadvantages which limit its consumption and its use. Among these are negative health aspects due to its high concentration of saturated fatty acids and cholesterol and its high price compared to vegetable fats. Furthermore, the chemical composition of milk fat varies across different regions and seasons. These natural fluctuations, primarily due to feeding practices, result in milk fats with different physicochemical properties (Jensen et al., 1991). In addition, the functionality of milk fat is often worse than that of vegetable fats such as margarines, restricting the potential use of milk fat in different food products such as puff pastry, cookies, and cold-spreadable products (Deffense, 1993). On the other hand, the much-appreciated flavour of AMF cannot be realized using these tailor-made vegetable products. Even addition of buttery aromas (e.g. diacetyl) seems unsatisfactory (Boudreau and Arul, 1993; Rajah, 1994). The utilization range of milk fat in many food applications could be broadened by the application of various modification processes, such as hydrogenation, interesterification and fractionation. These modification techniques alter the chemical composition of milk fat, and therefore the physical and nutritional characteristics, making milk fat more suitable in a wider field of application or for tailored specific end uses (Kaylegian, 1999; Van Aken et al., 1999). In this part, we will discuss the technology of milk fat fractionation and interesterification, and the influence of process parameters in more detail. The effect of these modification techniques on the composition and the physicochemical properties of milk fat is also covered. Hydrogenation of milk fat will not be considered here as this modification technique is no longer applied in the dairy industry. This is because the increased levels of saturated and trans fatty acids in hydrogenated milk fat are not desirable from a nutritional point of view. Also, the quite high production costs and the negative attitude of consumers towards chemical modification of a fat with a natural image militate against its commercial application. Therefore, milk fat hydrogenation is replaced industrially by other modification processes (especially fractionation) for the production of milk fat with an increasing hardness, oxidative stability, and plastic range (De Greyt and Kellens, 2001; Timms, 2005). 4.3.2 Fractionation Milk fat is composed of approximately 400 fatty acids and can thus contain, at the most, 4003 triglycerides. Due to this very heterogeneous composition, milk fat exhibits a wide melting range, from about −40°C to 40°C, which provides the possibility of separating milk fat into a series of fractions having their own physical and chemical properties. It has been accepted that the melting profile of natural milk fat contains three major peaks, due

74

Dairy-derived ingredients

Heat flow (W/g) exotherm up

0

–60

Fig. 4.1

–0.1 LMF MMF HMF –0.2 –0.3 Original milk fat –0.4 Olein Stearin –40

–0.5 –0.6 –20 0 20 Temperature (°C)

40

60

Melting profile of milk fat, stearin and olein determined with a heating rate of 5°C/min (Vanhoutte et al., 2002a).

to three major groups of triglycerides melting independently (Fig. 4.1): a low-melting fraction (LMF) which melts below 10°C, a middle-melting fraction (MMF) which melts from 10°C to 19°C, and a broad high-melting fraction (HMF) which melts above 20°C (Deffense, 1993; Kaylegian and Lindsay, 1995). Fractionation is the separation process of a fat into a higher melting solid fraction, the so-called ‘stearin’ and a lower melting liquid fraction, the so-called ‘olein’. The olein fraction is enriched in short-chain triglycerides and long-chain unsaturated triglycerides, while the ‘stearin’ fraction contains more long-chain saturated triglycerides. Deffense (1993) and Vanhoutte et al. (2002a) demonstrated that, in the melting curve of the milk fat olein fraction, the peaks of LMF and MMF enlarge, while HMF is completely removed at a fractionation temperature of 18°C. Conversely, the stearin fraction is enriched in HMF and contains less MMF (Fig. 4.1). The increase of LMF in the high-melting fraction is explained by olein entrainment in the solid fraction, due to incomplete separation. Fractionation of AMF is extensively reviewed by Kaylegian and Lindsay (1995) and, recently, more briefly by Augustin and Versteeg (2006). Different types of fractionation technologies have been employed and these technologies can be divided into two general groups: (i)

Fractionation methods based on crystallization and solubility behaviour of milk fat, including • Crystallization from melted milk fat, also known as dry fractionation (Deffense, 1993; Kellens, 1994; Lopez et al., 2006; Tirtiaux, 1976) or as Lanza fractionation in the case that a detergent solution is used to facilitate the separation of the fractions (Jebson, 1970).

Processing means for milk fat fractionation and production

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•

Crystallization from milk fat dissolved in a solvent, also called wet fractionation (Boudreau and Arul, 1993; Hartel, 2001; Larsen and Samuelsson, 1979; Marangoni and Lencki, 1998). (ii) Fractionation methods based on the solubility and volatility of the lipid constituents, including supercritical fluid fractionation (Kaufmann et al., 1982; Arul et al., 1987; Bhaskar et al., 1993) and short-path distillation (Arul et al., 1988; Campos et al., 2003). Dry fractionation Crystallization from melted fat (or dry fractionation) is the most commonly employed and commercially exploited fractionation technique. This technique is very attractive for the dairy industry due to its reasonable cost of processing and its relatively simple equipment. Other advantages of dry fractionation are the preservation of the delicate flavour of milk fat, the absence of organic solvents or other processing aids, and the environmentally-friendly character confirming the ‘natural’ product image (Fatouh et al., 2003). Today about 1000 tons per day of milk fat are fractionated with this fractionation process, predominantly in Europe. Dry fractionation consists of the development of suitable sizes of crystals by controlled cooling and agitating of the molten fat, followed by the separation of the crystals from the remaining mother liquor by filtration or centrifugation. The chemical composition and physical characteristics of the fractions obtained are significantly affected by the equipment design, the related fractionation process, separation method and other process parameters such as the cooling and agitation rate, the residence time of the fat in the crystallizer and the fractionation temperature (Grall and Hartel, 1992). Currently, dry fractionation is commercially executed by two different processes, the Tirtiaux (Deffense, 1993; Tirtiaux, 1976) and the De Smet (Kellens, 1994) processes, both from Belgium. Both processes start with heating the milk fat to a temperature of 60–80°C to ensure that all the nuclei are destroyed. The Tirtiaux and De Smet process differ in the way the cooling step is carried out. Tirtiaux has patented a slow-cooling process, usually carried out in vessels with gentle agitating. The fat is cooled following a special cooling pattern based on a constant temperature difference between the oil and the cooling liquid. Typically, the cooling rate is considerably decreased near the melting point, resulting in the formation of the desired β′ crystals, which are easier to separate. The first cooling step with the formation of crystal nuclei takes place in special buffer tanks. From there, the oil is pumped into crystallizers in which the cooling is regulated by constant monitoring of the temperature of the milk fat in the crystallizer. The intention of this second step is the growth of very large, equally-sized crystals (optimal size of 200 to 300 µm) which are easy to separate from the oil slurry. The agitation rate can also be adjusted in order to obtain crystals of the desired size and shape. The major shortcoming of this process is the

76

Dairy-derived ingredients

requirement of long crystallizing times (about 16 h to 20 h) to achieve the optimum crystallization. In contrast, the cooling in the De Smet process is also carefully monitored but it is applied in a different way. The processes recommended by De Smet follow the principle of fast cooling by lowering stepwise the temperature of the cooling liquid in precise and precalculated steps. In that way, the milk fat is precooled for sufficient crystal nuclei formation and the final fractionation temperature is reached more quickly. Due to this cooling program, a high cooling surface per unit volume of fat is necessary in the industrial design of the concentric crystallizers of the De Smet process. Additionally, filters and centrifuges with a higher separation efficiency compared to those applied in the Tirtiaux process are necessary as the fat crystals in the De Smet process are much more heterogeneous in size, resulting in a considerable higher filter cost. However, the De Smet process achieves its optimum crystallization in much less time (6 h to 8 h) than the Tirtiaux process. The next stage in this modification process is the separation step, resulting in the actual production of a solid product and a liquid fraction. The efficiency of this step has an enormous impact on the properties and yields of the resulting fractions. In the dairy industry, separation of the milk fat slurry is mostly accomplished by filtration. There are two different filtration techniques used; vacuum filtration on belt or drum filters and membrane press filtration. Vacuum filtration separates the liquid first by gravity and then the entrained olein is further removed under vacuum pressure. Although the separation can be carried out continuously, this technique does not permit a very thorough separation and a considerable amount of the olein fraction stays entrained in the filter cake. Classically, a continuous belt Florentine vacuum (50 to 200 mbar) filter, shown in Fig. 4.2, can be used in the Tirtiaux process since very large, equally-sized crystals are formed, which are easy to separate. In this type of filter, filtration takes place on the upper, horizontal surface of a rotating, stainless-steel perforated belt, which has a vacuum chamber beneath it (Illingworth, 2002). Better separation efficiency is achieved by using membrane press filters, introduced during the eighties in the fractionation industry. The application of pressure to the cake in the press chamber brings about a lower level of liquid entrainment in the filter cake. The higher pressure in the chambers of the membrane filter is generated by inflating the watertight membranes with air, reducing the chamber volumes. After deflating the membranes, the filter cakes can be batch-wise discharged into a tank by gravity. These principles of operation are illustrated in Fig. 4.3. Today, the membrane filter press is the most suitable filtration technique in milk fat dry fractionation and its usage is growing increasingly for all the crystallization processes. Especially in the case of the De Smet crystallization process, membrane filters are much preferred. Standard membrane filter presses normally operate at a maximum pressure of 4–8 bars, which is more than

Processing means for milk fat fractionation and production

77

Feed

Recycling

Air conditioning

Filter belt

Vacuum Scraping Blowing

Heating

Olein Stearin

Hot water system Steam

Fig. 4.2 A schematic diagram of a continuous belt Florentine vacuum filter (Illingworth, 2002).

Filling

Squeezing

Feed

Stearin concentration

Olein outlet

Fig. 4.3

Air inlet

Intersection of membrane plates during filling and squeezing (Calliauw, 2008).

78

Dairy-derived ingredients

sufficient in most cases. During recent years, new high-pressure membrane filter presses have been developed to operate at pressures of up to 30 bar. Those high-pressure membrane filter presses make use of hydraulic fluid as a pressure medium instead of air used for the standard membrane filter presses (Gibon and Tirtiaux, 2002). Higher pressures are necessary to meet the required specifications, especially in the production of high-purity olein fractions. Milk fat fractions can also be separated by use of centrifugal forces such as with nozzle and screen centrifuges. As yet, only a few commercial installations are currently known to exist. For instance, Westfalia developed and launched in 1997 a nozzle centrifuge as an alternative to membrane filtration for AMF fractionation. In this piece of equipment, the fat crystals are separated in a centrifugal field due to the density difference of olein and stearin. During the operation, crystals are drawn outwards where they are discharged through backwardly reacting nozzles by the centrifugal force. This type of separation can be done continuously and in an enclosed system, the latter being very important in preventing the possible deterioration of milk fat (fractions) when exposed to oxygen or light. However, as stated, only a few installations have been used on an industrial scale, possibly due to a separation efficiency which is not as good as those of the membrane press, and due to complicated operation handling operations (Deffense, 2000; Dijkstra, 2007; Illingworth, 2002). The fractionation process, and more predominantly the crystallization and filtration properties, are greatly affected by a number of process parameters such as agitation rate, cooling rate and fractionation temperature. The effect of these parameters on milk fat fractionation and the properties of the resulting fractions have been investigated several times (Deffense, 1987; Patience et al., 1999; Vanhoutte et al., 2002a, 2003). For instance, Vanhoutte et al. (2003) concluded that higher fractionation temperatures result in a lower stearin yield and a higher melting point of the olein fraction. Fractionation temperature also influences the cake density and thus the filtration characteristics to a certain extent, probably related to differences in crystal size and/or morphology. At lower temperatures, more compacted clumps of smaller crystals are formed, resulting in less oil entrapment and a denser filter cake. The agitation rate also has a considerable impact on the crystal size and/or morphology and thus on the filtration behaviour. Herrera and Hartel (2000) noticed that samples crystallized at the highest shear rates will produce the narrowest particle size distribution and the smallest crystals, as a result of an enhanced nucleation rate (Herrera and Hartel, 2000) and breakage of the crystals by the shear forces of the impeller (Patience et al., 1999). On the other hand, agitation ensures effective heat transfer on the cooled surfaces of the crystallization vessel and avoids local overheating and subsequent melting of the crystals. Some minor components of the milk fat can also alter the fractionation properties. Vanhoutte et al. (2002b) observed that high levels of free fatty

Processing means for milk fat fractionation and production

79

acids (from inadequate heat treatment or microbial contamination) or high levels of partial glycerides (from a deodorization process) have a negative influence on the filtration quality of the resulting crystal suspension. In the small-scale fractionation experiments, lower filtration rates and an increased oil inclusion resulting in a softer stearin fraction were detected. The presence of phospholipids changes negatively both the crystallization kinetics and the filtration properties. Increased induction times and worse filtration qualities leading also to a softer stearin, were observed at higher concentrations of added phospholipids. These results emphasise the importance of good quality raw materials for fractionation of AMF and an adequate removal of the membranous material (containing a lot of phospholipids) during the production of AMF. By fractionation, two fractions are produced from the original milk fat, with modified chemical and physical properties. However, for applications in a broad range for food products, the separation of AMF into sharply defined fractions usually happens in a multi-stage process. In such a multistage process, one or both of the fractions are remelted and refractionated but with different process parameters and cooling patterns. Most commonly, the olein of the first step is fractionated again at a fractionation temperature that is lower than the temperature of the first step. This process can then be repeated. In the dairy industry, most of the fractionation processes of AMF happen in three steps, although seven steps have been reported in literature. Figure 4.4 presents some fractionation routes applied to milk fat, together with the melting characteristics of the fractions obtained (Deffense, 1993; Illingworth, 2002; Kaylegian, 1999; Kaylegian and Lindsay, 1995).

Third stearin: OSS DP 32°C Hard stearin: S DP 45°C AMF DP 34°C

Second (soft) stearin: OS DP 26°C

T21°C-22°C Olein: O DP 21°C

Olein: OSO DP 24°C T 12°C-15°C Stearin: OSS DP 21°C Super olein: OO DP 13°C

TC42). Despite the use of 1,3-specific lipases, no positional specificity was observed in the different studies. This can probably be explained by the non-enzymatic acyl migration which is a migration of the fatty acids to the neighbour position on the glycerol backbone resulting in a redistribution of the fatty acids at the sn-2 position as well. This changed triglyceride profile has a considerable effect on the melting characteristics of the interesterified fat. Enzymatic interesterification also results in a decrease of the solid fat content at lower temperatures, and above 25°C it generally leads to an increase. This smoother, more linearized SFC-profile was also observed by Marangoni and Rousseau (1998), although they found that the SFC drops upon enzymatic interesterification at all temperatures. Lower SFC values at every temperature may result from an increase in water content in the reaction medium yielding higher amounts of mono- and diglycerides (Kalo et al., 1986). Compared with chemical interesterification, the enzymatic catalyzed process has a lower impact on the typical flavour components of milk fat. Another major benefit is its qualification as a ‘green’ chemistry solution due to lower amount of product losses and waste products. The milder reaction condition also leads to a more environmentally friendly process. On the other hand, the biocatalyst costs more and requires a longer reaction time and better reaction control, which may result in increased processing costs (De Greyt and Kellens, 2001). Applications of interesterified milk fat The modifications of the physical properties creates the possibility of using interesterified milk fat in a wider range of food products. For instance, Timms and Parekh (1980) reported that interesterified milk fat seems to be better suited to chocolate than unmodified milk fat. Moreover, Huyghebaert et al. (1994) stated that the texture of recombined butter made from interesterified milk fat has a better spreadability at 5°C than the original butter. However, the product is not fully spreadable and can be further improved by removing the high melting triglycerides by fractionation. Neither chemical nor enzymatic interesterification of milk fat has yet been carried out on a commercial scale. The major drawback for its industrial exploitation is the high operating cost, which is mainly the result of considerable oil losses. The process is commercially viable only if this high operating cost is offset against the added value of the interesterified product. In the case of milk fat, this seems very doubtful, because interesterification has a negative impact on the desirable buttery flavour (De Greyt and Kellens, 2001).

86

4.4

Dairy-derived ingredients

Milk fat minor components

4.4.1 Downstream processing of polar lipids The polar lipids in dairy products, which comprise PLs and sphingolipids, are predominantly situated in the MFGM membrane (60–70%) and the lipoprotein particles. The MFGM membrane surrounds the fat globules, stabilizes the fat droplets in the milk serum, and protects them from enzymatic degradation by lipases (Danthine et al., 2000). During the processing of milk (e.g. churning), destabilization of the fat globules and disruption of the MFGM occurs. Then this biological membrane becomes no longer associated with the fat globules and the MFGM fragments preferentially migrate to the aqueous phases such as buttermilk and butter serum (McPherson and Kitchen, 1983). As the polar lipids of the MFGM are closely associated with a variety of specific membrane proteins, of which xanthin oxidase/dehydrogenase, butyrophilin and adipophilin are the most abundant (Mather, 2000), the MFGM proteins will probably co-migrate with the polar lipids during dairy processing. Corredig et al. (2003), Roesch et al. (2004) and Rombaut et al. (2007) have confirmed that dairy products with a high polar lipid content are also enriched in MFGM proteins. Generally, the most promising dairy products for further purification of MFGM components are products with a high polar lipid content on either a dry matter or a total lipid base (e.g. buttermilk, butterserum and whey), because water can easily be removed by filtration, evaporation or drying. Nonetheless, solvents for extraction and fractionation are often required to separate neutral and polar lipids. Various techniques have been used for purification of MFGM fragments, such as coagulation, filtration, centrifugation and conventional or supercritical extraction techniques or combinations thereof (Rombaut and Dewettinck, 2006). Laboratory applications for MFGM isolation Generally, laboratory MFGM isolation methods consist of four major steps (Singh, 2006), namely the separation of the fat globules by centrifugation, followed by several washing steps to remove contaminants (Mangino and Brunner, 1975; Kanno and Kim, 1990; Ye et al., 2002; Fong et al., 2007), the disruption of the fat globules to release the MFGM and finally, the recovery of the MFGM material. An overview of the various washing solutions that have been used for MFGM isolation is given in the review of Dewettinck et al. (2008). Churning, agitation at reduced temperatures, freeze–thaw cycles, extractions with polar aprotic solvents, bile salts, or nonionic detergents are often used to release the MFGM from the triglyceride fat core from the washed fat globules into the aqueous phase (Dowben et al., 1967; Harrison et al., 1975; Snow et al., 1980; Patton, 1982; Dapper et al., 1987). The final step involves the collection of the released MFGM material by ultracentrifugation, freeze-drying or microfiltration. The membrane material can also be obtained by precipitation at low pH or by ‘salting out’ with

Processing means for milk fat fractionation and production

87

ammonium sulfate and recovering the precipitated material by centrifugation (Anderson and Brooker, 1974; Kanno and Kim, 1990; Rombaut et al., 2006a; Morin et al., 2007a). The above mentioned procedures are laboratory applications for the isolation of MFGM material from untreated milk, which are summarized in Fig. 4.5. MFGM isolation from industrial sources Rich sources of MFGM material such as buttermilk, butter serum and whey are still considered as low value by-products originating from dairy processing. Generally, these products have a low stability to oxidation due to their higher polyunsaturated fatty acid content, and a high variability between batches. Furthermore, the processing steps affect the interactions of the serum proteins with the MFGM. The lack of whey proteins recovered in the permeate illustrates the strong binding of whey proteins with MFGM components. The resulting aggregates are too large to pass through the membrane pores during microfiltration and are recovered in the retentate (Ward et al., 2006). Corredig and Dalgleish (1997) and Morin et al. (2007b) showed that these interactions most likely to occur during heating the MFGM isolates, even at temperatures as low as 65°C. However, to date, only limited data are available on the influence of processing on the MFGMinteractions, so more research should be undertaken to better understand them and to control the manufacturing conditions to obtain products of consistent quality. In spite of this, many authors have used buttermilk as a raw material for the isolation of MFGM fragments. Tangential micro- and ultrafiltration techniques seem to be appropriate for MFGM purification from dairy byproducts, because of their ability to separate particles from dissolved solutes. Filtration conditions such as pH, temperature, type of membrane material, pore size and the type of buttermilk will influence the permeation of the different components of the buttermilk through the membrane (Morin et al., 2004, 2006; Rombaut et al., 2007). The isolation of polar lipids by membrane filtration is based on the selective removal of casein, whey proteins, lactose and minerals from the concentrate. Sometimes the filtration is combined with the stepwise addition of water, for further washing out of undesirable components such as lactose, whey-proteins and minerals (¼ diafiltration). However, microfiltration alone cannot achieve optimal separation of MFGM lipids and proteins from caseins and whey proteins. Buttermilk and whey are rich sources of MFGM material, but they also may contain significant amounts of skim milk components, such as whey proteins and caseins. The casein micelles can cause difficulties during the purification of polar lipids by cross flow membrane filtration. They have sizes similar to the MFGM fragments and filtration solely cannot remove these micelles. Sachdeva and Buchheim (1997) achieved a tenfold increase in polar lipid concentration (70–77% recovery) by removing the caseins by acid and rennet coagulation prior to filtration. Another strategy is reduction

88

Dairy-derived ingredients Milk (Re)separate fat 2 - 4x

Cream

Washing solution

Skimmed milk Discharged washing solution

Wash Washed cream - Churning

Release MFGM - Freezing and thawing Extract MFGM - ...

Separate

- Polar aprotic solvents - Bile salts - Nonionic detergents

Centrifuge Buttermilk

Butter Melt Centrifuge Butter oil

Combine

Butter serum

MFGM suspension

Permeate

Filtrate (membrane)

Ultracentrifuge

MFGM isolate

MFGM pellet Freeze dry

MFGM supernatant Freeze dry

MFGM

Defat (solvent) MFGM protein Dialysis Freeze dry

Pure MFGM protein

Fig. 4.5

Summary of isolation methods of MFGM (Dewettinck et al., 2008).

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of the casein micelle diameter, which allows it to be transferred through the membrane and recovered in the permeate. Corredig et al. (2003) and Roesch et al. (2004) used sodium citrate to dissociate the casein micelles into submicelles, followed by microfiltration to collect MFGM material in the retentate. In this way, the fat and casein content were increased in the retentate and permeate respectively. An enriched fraction from buttermilk containing about 80% of MFGM material with a significantly reduced amount of caseins was obtained (Corredig et al., 2003). The application of several diafiltration steps with deionized water resulted in a reduction of the casein contamination in the retentate, but also in a loss of MFGM material (Rombaut et al., 2006a). Roesch and Corredig (2002) described an enzymatic treatment with trypsin and chymotrypsin to destroy the casein micelles. This procedure reduced the retention of caseins during microfiltration, leading to a phospholipid-enriched retentate. Rombaut et al. (2006a) added citrate to butter serum. These authors reported that 44% of the polar lipids were lost during filtration due to blocking and fouling of the filter membrane with MFGM particles. Addition of sodium citrate agent caused dispersion of not only casein micelles but also of MFGM fragments. Morin et al. (2006) compared the fractionation of regular buttermilk and whey buttermilk, the aqueous fraction obtained by churning of whey cream which is separated from cheese whey, by ceramic microfiltration. The latter contains similar amounts of PLs to regular buttermilk and the absence of caseins helps to minimize separation problems. However, the variability of the lipid content of whey cream results in considerable variations in the lipid content of whey buttermilk. Careful standardization of the whey cream prior to churning should be considered. Rombaut et al. (2007) used acid buttermilk cheese whey, the aqueous fraction obtained by acidification of sweet-cream buttermilk, as a starting material for concentration of MFGM components by microfiltration. Acid buttermilk cheese whey is relatively rich in MFGM fragments and, as in whey buttermilk, the casein micelles are absent. The authors reported that the membrane material and pore size have a great influence on filtration characteristics and MFGM retention. The best results were obtained with negatively charged membranes with a pore size less than 0.15 µm to avoid excessive MFGM losses. MFGM retention was also affected by the pH and at optimized conditions, 98% of the polar lipids from the acid buttermilk whey could be recovered in the retentate. A thermocalcic aggregation (* see page 90) whey pre-treatment for whey clarification resulted in low permeate fluxes and high retention of ash and whey proteins, which makes it less suitable as a MFGM purification technique. Another approach is the use of cream washed with skim milk ultrafiltrate to remove the caseins prior to churning, which results in a protein reduction of 74% compared with normal cream buttermilk (Morin et al., 2007a). This procedure induced losses of MFGM material and a yield reduction of the

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process, but it significantly improved the permeation flux, the concentration of MFGM material and the removal of skim milk proteins. *Thermocalcic aggregation is a sequestration technique of residual lipids, lipoproteins, and MFGM fragments; it was originally developed for the clarification of sweet cheese whey. After the coagulation of the polar lipid components by calcium addition and moderate heating, these aggregates can be separated by settling, filtration, or centrifugation. Thermocalcic aggregation is often applied in the manufacture of whey protein concentrates as a pre-treatment prior to UF, because the residual fat, consisting mainly of small fat globules, liposomes and MFGM particles, would lead to rapid fouling and flux decline of the membranes. Around 80% of the β-lactoglobulin and 85% of the α-lactalbumin remain soluble in the clarified whey, and the resulting sediment contains merely all polar lipids (Fauquant et al., 1985; Pierre et al., 1992). Theodet and Gandemer (1994) concentrated the polar lipids of Emmenthal whey by thermocalcic aggregation followed by microfiltration, and reported an increase in polar lipid concentration from 0.33 g to 1.10 g/100 g dry matter. Rombaut et al. (2006b) observed a similar rate of increase of 1.84 g to 7.95 g/100 g dry matter using buttermilk whey instead of Emmenthal whey. The combination of membrane techniques, thermocalcic aggregation, enzymatic hydrolysis and demineralization, results in much higher degrees of purification, as observed by Baumy et al. (1990). These authors obtained a concentrate containing 16.79 g/100 g dry matter starting from whey, and combined with solvent extraction and precipitation even higher concentrations were reported. The hexane–isopropanol extraction resulted in a polar lipid concentration of 36.8 g/100 g dry matter, whereas subsequent acetone precipitation yielded 76.7 g/100 g dry matter. In general, the coagulation and filtration techniques do not seriously alter the polar lipid/total lipid ratio. These processes do not exhibit selective effects on lipids, so solvent extraction and fractionation techniques are required to increase this ratio. Supercritical CO2 (SCO2) is excellently suited as an extraction solvent due to its low critical parameters (31.1°C, 73.8 bar), relatively low cost, non-toxicity, chemical inertness and nonflammability. Furthermore, the resulting products are free from organic solvent residues. It is therefore an environmentally benign alternative to conventional lipid fractionation techniques, based on toxic solvents. Supercritical fluid extraction can be used to extract lipid and lipid-soluble materials from complex matrices. Astaire et al. (2003) developed a two-step procedure to increase concentrations of the polar MFGM lipids by microfiltration and supercritical fluid extraction (SFE) from buttermilk. They removed the triglycerides present in microfiltration-processed buttermilk powder using SCO2 extraction, thereby increasing the polar lipid content from 31.02 g to 83.15 g/100 g total lipids. The resulting MFGM isolate contained a significantly reduced concentration of non-polar lipids and an increased concentration of polar lipids derived from the MFGM. Most

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likely, the inclusion of a second SCO2 extraction step, with addition of a modifier such as ethanol, would exclusively remove the polar lipids, thereby resulting in a 100% pure fraction. This was carried out on ground soybean by Montanari et al. (1999). A first SCO2 extraction step of soybean flakes was performed to extract the oil, leaving the PLs in the defatted soybean flakes, after which the PLs were recovered by a second SCO2/ethanol extraction step resulting in a PL rich fraction. The PLs will not effectively dissolve in CO2, and thus a suitable co-solvent such as ethanol is added to enhance their solubility. These results are remarkable, as the traditional extraction with hexane leaves about 50% of the total PLs in the spent seed matrix, while the other 50% are extracted into the hexane.

4.4.2 Technological functionalities PLs make up approximately 1% of milk lipids, and they play an important role in the milk structural system. PLs are amphiphilic, and in milk they are present mostly in the MFGM, where they stabilize the fat globule against coalescence. Due to their dipolar nature, PLs in general, and milk PLs in particular, are considered to have good emulsifying properties. The forms of milk PLs used in the food industry have evolved from byproducts of the dairy processing industry which have a high content of MFGM to the use of isolated and fractionated ingredients which are concentrated in the PL content. Compared to skim milk powder, buttermilk powder has better emulsifying properties since it contains a higher concentration of PL. Because of that, the former has been being replaced by the latter in the bakery industry. Buttermilk is also used to replace partially skim milk in dairy products such as yoghurt and cheese due to its lower cost and nutritional properties. Adding a certain amount of liquid buttermilk, which is concentrated by several means, reduces the free oil under baking and increases the yield of pizza cheese by increase moisture content without adversely affect functional properties of the cheese (GovindasamyLucey et al., 2007; Mistry et al., 1996). Similar findings were observed with production of Mozzarella cheese (Poduval and Mistry, 1999). Whey buttermilk, resulting from churning of whey cream in the manufacture of whey butter, gave higher emulsifying properties and lower foaming ability compared with sweet or cultured buttermilk, probably due to a higher ratio of PL to protein (Sodini et al., 2006). MFGM isolated from untreated milk, which contains 25–30% PL depending on the isolation methods, has a good emulsifying capacity. One percentage of MFGM material isolated from untreated milk is enough to stabilize an emulsion made from 25% milk fat and the emulsion stability increases with increasing concentration of the MFGM (Kanno, 1989). The fat globule size ranges from 0.9–17 µm in diameter (Kanno et al., 1991). The effects of emulsifying time, homogenization, temperature, and pH on emulsifying properties, size and the surface of globules were also investigated in

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this study. The emulsion had the highest viscosity and was the least stable at pH around 5, which is near the isoelectric point of MFGM, 4.9 (Kanno, 1989; Kanno et al., 1991). However, the emulsifying properties of MFGM material isolated from industrial buttermilk are not always superior compared to that of the buttermilk (Corredig and Dalgleish, 1997; Wong and Kitts, 2003b). This varying observation on emulsifying properties has been attributed to the difference in protein composition of the isolated MFGM material. Attachment of β-lactoglobulin, and to a lesser extent α-lactalbumin and κ-casein, to the MFGM during heating of milk is well-known (Ye et al., 2004). As a result, MFGM material isolated from industrial sources is different in composition compared to that of the material isolated from untreated milk. Along with proteins, PLs contribute a major part of the milk fat globule membrane. However, there is limited information available on the effect of processing treatments on this moiety. Whether or not the changes in PL composition or chemical structure, or alteration in interaction of PLs together or with other components of the membrane or of the milk serum phase, would change the technological properties of the MFGM isolate is still to be investigated. Corredig and Dalgleish (1997) concluded that heating the MFGM isolate strongly affected the technological properties of these membrane fractions, caused by association of the whey proteins with the MFGM. Using capillary electrophoresis, bovine serum albumin and other proteins were shown to interact with PS and PC when they were incubated together (Bo and Pawliszyn, 2006; Hu et al., 2001). MFGM material isolated from raw milk is currently used mainly for research purposes while potential applications today relate to the enriched PL fractions isolated from the industrial sources. As summarized in the previous section, many researchers have been successful in isolating MFGM fragments, which contain high concentrations of PLs and specific membrane proteins, from by-products of dairy industrial processing. This opens opportunities to make use of such cheap sources to manufacture addedvalue ingredients with special functionalities. However, the treatment during milk processing affects the composition of the materials obtained as well as their technological functionalities. Further studies are still needed on this aspect and certain adaptation of dairy processing procedures would probably be needed in order to protect technological and nutritional properties of the MFGM which is concentrated in the by-products after processing of milk or cream (Ward et al., 2006). Soybean and egg yolk lecithin have a long tradition of being employed in food products, as well as others such as cosmetic products, animal feeds, medicinal and agrochemical products, paints and lubricants. However, the use of PL from dairy sources is limited to pharmaceutical and cosmetic applications due to technological difficulties in extracting and hence high prices of the prepared isolates (Miura et al., 2006). Soy lecithin does not contain SM and contains very limited concentrations of PS, while PL fractionated from dairy sources contains about 24–32% SM and 3.2–12% PS

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(Burling and Graverholt, 2008; Miura et al., 2006; Thompson and Singh, 2006). The cost issue which limits applications of bovine milk lecithin would become less important as its beneficial properties in both technological as well as nutritional aspects are fully accounted. Several dairy functional ingredients enriched in PLs have been launched on the market. Lacprodan-20 and -75 from Arla Foods Ingredients amba (Denmark) contain 20 and 75% PLs, respectively, and Phospholac 600, produced by Fonterra Cooperative Group Ltd. (New Zealand) contains more than 70% of PLs. One interesting result from the use of such enriched PL isolate has been reported from the research group of Snow Brand Milk Products (Japan), where bovine milk lecithin (85% PL) was found to stabilize while soy lecithin (95% PL) solidified the cream reconstituted from butter oil (Miura et al., 2006). Among the PL species, PC but not PE or SM, regardless of their origin, is the component to exert the emulsifying effect (Miura et al., 2004, 2006). Several forms of liposomes, unilamellar, multilamellar, and multivesicular, can be created from Phospholac 600 using microfluidization, and these PL dispersions are quite stable towards oxidation (Thompson and Singh, 2006). The capacity to produce liposomal formulations gives rise to opportunities to use MFGM PLs to improve moisturizing effects, and to encapsulate and protect sensitive cosmetic ingredients or bioactive ingredients in functional foods. The capacity of SM to improve the epidermal function is one of great attraction for employing milk PLs in cosmetic and pharmaceutical applications (Haruta et al., 2008). Compared to triglycerides, PLs of milk contain a high concentration of unsaturated fatty acids, so they are very susceptible to oxidation. The rate of milk fat oxidation is well-known to be accelerated in the presence of oxidizing factors such as oxygen, light, heat and catalysts such as metal ions. Oxidation is, in almost all cases, an important issue to deal with when phospholipid-rich ingredients are used. However, PLs can be pro-oxidative or anti-oxidative to milk fat, depending on processing conditions and intrinsic factors of the matrices where PLs are incorporated (Chen and Nawar, 1991a,b; King, 1963; Lee and Choe, 2008). The antioxidation ability of PLs depends both on their head groups and their fatty acid composition (Nwosu et al., 1997). An amount of 0.1–0.2% buttermilk powder inhibits lipid oxidation in a simple peroxidizing model system and the reducing activity of the buttermilk is mainly attributed to sulfhydryl groups (of proteins). These groups may scavenge peroxide and hydroxyl radicals, and sequester both Fe2+ and Fe3+ (Wong and Kitts, 2003a). The nutritional properties of PL will be highlighted in the following section. For applications of milk PL, or dairy ingredients enriched in PL, in functional foods or any other products, one could utilize their technological functionalities, beneficial nutritional properties or their protective effects (e.g. in the form of liposome) or any combination of these three advantages.

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Nutritional properties of dairy fats

A large amount of research data with focus on the nutritional properties of dairy lipids is now available. Nevertheless, additional data are still needed to gain better insights in the role of dairy lipids in nutrition and health, and to make appropriate recommendations for consumers in terms of dairy fat intake, and to producers for modification of dairy lipid composition and production of modified food products. Currently, the nutrition debate on dairy lipids has a high focus on the relationship between dairy fat intake and cardiovascular disease (Steijns, 2008; Tholstrup, 2006). The ratio total cholesterol : HDL cholesterol in serum is now considered as a more sensitive and specific risk factor for coronary heart disease (CHD) compared with total cholesterol (Mensink et al., 2003). CHD risk is lowered when saturated fatty acids (SFAs) and trans-monounsaturated fatty acids are replaced by cis-unsaturated fatty acids. In the case of individual SFAs, myristic acid (C14 : 0) and palmitic acid (C16 : 0) show limited change in total cholesterol : HDL cholesterol when replacing 1 E% from carbohydrate, and are considered to have a negative effect on CHD risk (Mensink et al., 2003; Steijns, 2008). Although myristic and palmitic acids as well as trans-monounsaturated fatty acids are important constituents of dairy fats, their effects on CHD risk when present in ingested dairy products are still far from understood. Milk fat consumption shows less pronounced effects on serum lipids than expected from its fat content, and epidemiological data frequently lack an association between milk consumption and CHD. Emerging issues that receive or need further attention in CHD research are: (i) the role of short and medium chain fatty acids and sphingolipids in milk and milk products; (ii) the influence of specific non-fat components in milk and milk products, e.g. calcium, proteins, peptides and vitamin K; (iii) the type of dairy product used to carry dairy fat, e.g. cheese consumption may have more favourable effects on serum lipoprotein levels compared to butter at equal saturated fat intake; (iv) the influence of stereospecific distribution of fatty acids in triglycerides; and (v) the influence of dairy constituents on additional biomarkers for CHD risk, e.g. small and dense LDL particles (Pfeuffer and Schrezenmeir, 2006; Haug et al., 2007; Tholstrup, 2006; Steijns, 2008). A more complete scientific update on the relationship between dairy fats and cardiovascular disease has recently been published (Lock et al., 2008). Next to effects on cardiovascular disease, effects on other disorders are also being evaluated, such as insulin resistance and type 2 diabetes, obesity, and cancer (for reviews, see Mensink, 2006; Collomb et al., 2006; Pfeuffer and Schrezenmeir, 2006). The milk fat globule membrane in milk containing a high level of polar lipids has also received significant attention in view of its potential bioactive effects (for a review, see Dewettinck et al., 2008). For instance, it is believed that PC supports liver recovery from chemical or viral damages, and has a protective effect on the gastrointestinal mucosa

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against toxic attacks. Furthermore, some PLs are digested in the gastrointestinal tract to metabolic breakdown products that might possess antibacterial and antiviral activity.

4.6

Acknowledgement

The financial support of grant G.0490.08 from the Fund for Scientific Research – Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen) is gratefully acknowledged.

4.7

References

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marangoni a g and lencki r w (1998), ‘Ternary phase behavior of milk fat fractions’, J Agric Food Chem, 46, 3879–3884. DOI:10.1021/jf9801668. marangoni a g and rousseau d (1998), ‘Chemical and enzymatic modification of butterfat and butterfat–canola oil blends’, Food Res Int, 31, 595–599. DOI:10.1016/ S0963-9969(99)00033-2. mather i h (2000), ‘A review and proposed nomenclature for major proteins of the milk-fat globule membrane’, J Dairy Sci, 83, 203–247. mcpherson a v and kitchen b j (1983), ‘Review of the progress of dairy science – The bovine milk fat globule membrane – Its formation, composition, structure and behavior in milk and dairy products’, J Dairy Res, 50, 107–133. mensink r p, zock p l, kester a d and katan m b (2003), ‘Effects of dietary fatty acids and carbohydrates on the ratio of serum Total to HDL cholesterol and on serum lipids and apolipoproteins: A meta-analysis of 60 controlled trials’, Am J Clin Nutr, 77, 1146–1155. mensink r p (2006), ‘Dairy products and the risk to develop type 2 diabetes or cardiovascular disease’, Int Dairy J, 16, 1001–1004. DOI:10.1016/j.idairyj.2005.10.013. mistry v v, metzger l e and maubois j l (1996), ‘Use of ultrafiltered sweet buttermilk in the manufacture of reduced fat Cheddar cheese’, J Dairy Sci, 79, 1137–1145. miura s, mutoh t, shiinoki y and yoshioka t (2006), ‘Emulsifying properties of phospholipids in the reconstitution of cream using butter oil’, Eur J Lipid Sci Technol, 108, 898–903. DOI:10.1002/ejlt.200600109. miura s, tanaka m, suzuki a and sato k (2004), ‘Application of phospholipids extracted from bovine milk to the reconstitution of cream using butter oil’, J Am Oil Chem Soc, 81, 97–100. DOI:10.1007/s11746-004-0863-1. montanari l, fantozzi p, snyder j m and king j w (1999), ‘Selective extraction of phospholipids from soybeans with supercritical carbon dioxide and ethanol’, J Supercrit Fluids, 14, 87–93. DOI:10.1016/S0896-8446(98)00110-7. morin p, britten m, jiménez-flores r and pouliot y (2007a), ‘Microfiltration of buttermilk and washed cream buttermilk for concentration of milk fat globule membrane components’, J Dairy Sci, 90, 2132–2140. DOI:10.3168/jds.2006-832. morin p, jiménez-flores r and pouliot y (2004), ‘Effect of temperature and pore size on the fractionation of fresh and reconstituted buttermilk by microfiltration’, J Dairy Sci, 87, 267–273. morin p, jiménez-flores r and pouliot y (2007b), ‘Effect of processing on the composition and microstructure of buttermilk and its milk fat globule membranes’, Int Dairy J, 17, 1179–1187. DOI:10.1016/j.idairyj.2007.03.010. morin p, pouliot y and jiménez-flores r (2006), ‘A comparative study of the fractionation of regular buttermilk and whey buttermilk by microfiltration’, J Food Eng, 77, 521–528. DOI:10.1016/j.jfoodeng.2005.06.065. norris r, gray i k, mcdowell a k r and dolby r m (1971), ‘The chemical composition and physical properties of fractions of milk fat obtained by a commercial fractionation process’, J Dairy Res, 38, 79–82. nwosu c, boyd l and sheldon b (1997), ‘Effect of fatty acid composition of phospholipids on their antioxidant properties and activity index’, J Am Oil Chem Soc, 74, 293–297. DOI:10.1007/s11746-997-0139-4. palmquist d l, beaulieu a d and barbano d m (1993), ‘Feed and animal factors influencing milk fat composition’, J Dairy Sci, 76, 1753–1771. parks o w, schwartz d p, katz l and keeney m (1964), ‘Isolation and characterization of methyl ketone precursor in butter fat’, J Lipid Res, 5, 232–235. patience d b, hartel r w and illingworth d (1999), ‘Crystallization and pressure filtration of anhydrous milk fat: Mixing effects’, J Am Oil Chem Soc, 76, 585–594. DOI:10.1007/s11746-999-0008-4.

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5 Modern approaches to lactose production R. J. Durham, University of Western Sydney, Australia

Abstract: This chapter reviews recent improvements in the purification and crystallisation of lactose from whey. The properties of lactose and traditional methods of manufacture are discussed, within this framework methods for whey and permeate pretreatments are examined including; calcium precipitation, nanofiltration, ion exchange, electrodialysis, adsorptive decolourisation and ion exclusion chromatography. The fundamentals of lactose crystallisation are explored together with the impact of impurities on lactose crystal growth rate, purity, shape and size distribution. Innovations in lactose crystallisation and specialised pharmaceutical products are discussed including spray dried and anhydrous lactose, and dry powder inhalers. Post handling, separation, washing, milling and drying and impacts on lactose quality are described. Finally standards for hygiene and quality control of lactose manufacture, applications and functionality of lactose are covered in the last part of this chapter. Key words: lactose, production, manufacture, whey, calcium, nanofiltration, ion exchange, chromatography, crystallisation, pharmaceutical, edible.

5.1

Introduction

Lactose is the major carbohydrate found in the milk of mammals, whereby cows’ milk contains about 4.4–5.2% lactose and human milk 6.5–7.5% (Ganzle et al., 2008). Lactose is typically recovered from whey, a by-product from cheese or casein manufacture. Recovery has mostly relied on the low solubility of lactose, using crystallisation as the basis for separation and purification. Early methods involved crystallisation from concentrated whole whey, which may have been adjusted to pH 6.8 and boiled to precipitate whey proteins and calcium. It was concentrated to 60% solids in single-effect vacuum pans, then the lactose was allowed to crystallise. The resulting crude lactose crystals were then washed and dried in rotary driers

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(Weisberg, 1954). Purity and yields were low. Advances in membrane technology in the 1970s led to vast improvements in whey processing, with reductions in membrane costs throughout the 1980–1990s, enabling the efficient recovery of whey proteins as a valuable by-product from whey, creating protein-free permeates for lactose manufacture. Improvements in lactose manufacture have addressed purification as a key determinant of process efficiency, with research and development focused on calcium precipitation, nanofiltration, ion exchange, decolourisation and chromatography. Research on lactose crystallisation has been aimed at better understanding of crystallisation at the molecular level, for better control over lactose crystal purity, shape and size distribution. There has also been much research directed at understanding the anomeric character of lactose and transformations during drying, milling and storage, and consequences on lumping and post-handling. Finally, the development of sophisticated pharmaceutical lactose products, such as dry powder inhalers, has been the sharp edge of lactose research, directed at developments in particle engineering for fine control over lactose morphology and performance of lactose as a drug delivery device.

5.1.1 Properties of lactose Lactose is a disaccharide with a molecular formula of C12H22O11, composed of D-galactose and D-glucose linked by a β-1,4 glycosidic bond. The aldehyde group of the glucose moiety remains free and is able to be oxidised; therefore lactose is a reducing sugar (Rosenthal, 1991). Lactose has an anomeric centre at the C1 carbon of the glucose moiety, with the hydroxyl group able to be positioned in an equatorial (β-anomer) or axial (α-anomer) direction relative to the pyranose ring, see Fig. 5.1. Lactose in solution exists in dynamic equilibrium between α and β anomeric forms. Mutarotation rates are affected by pH, temperature, ionic concentration and solvent. The solubility of the α and β anomers differ

CH2OH O OH

CH2OH O OH OH

O OH

OH α-Lactose

CH2OH O OH OH

CH2OH O OH O

OH β-Lactose

Fig. 5.1 The chemical structure of the α- and β-lactose.

OH

OH

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significantly, for example the solubility of α-lactose monohydrate is 7 g/100 g water at 15°C, β anhydrous is 50 g/100 g water, while an equilibrium mixture of α and β is 17 g/100 g water at 15°C (Hartel and Shastry, 1991). The equilibrium lactose solubility increases with temperature and is determined by the mutarotation rate below 93.5°C. Above this temperature, β-lactose solubility determines the overall solubility. Equations describing the equilibrium solubility (g lactose/100g water) of anhydrous lactose were developed by Lifran 2007 (Eqs 5.1 and 5.2), using data by Hudson (1904), Herrington (1934), Foremost Foods (1970) and Visser (1982). Lactose solubility C S = e 2.389 + 0.028 T at T 93.5°C

[5.1] [5.2]

(Lifran, 2007) Lactose normally crystallises as α-lactose monohydrate, containing one molecule of water for each molecule of lactose. The crystals are very hard and dissolve slowly, and if their size is over 10–14 m µ , they can be detected sensorially, creating a defect known as sandiness (Ganzle et al., 2008). α-Lactose monohydrate crystals normally form a tomahawk shape during crystallisation (see Fig. 5.2). The tomahawk shape arises from inhibition of growth of the 0{ 11}face, by the attachment and inclusion of

–b K (~150)

010

110

110

011

011

100

100 110

–a

110

010

70 –c

Fig. 5.2 Tomahawk shape of α-lactose monohydrate crystals (van Kreveld and Michaels, 1965).

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β-lactose at this face inhibiting further growth (Dincer et al., 1999; Raghavan et al., 2000). β-lactose is the predominant crystalline form for crystallisations conducted above 93°C. Amorphous lactose forms when lactose is quickly dried and consists of a glassy state mixture of α and β lactose. Amorphous lactose is very hygroscopic, with a strong tendency to recrystallise into α-lactose monohydrate, forming hard lumps during storage. The properties of crystalline lactose are affected by its polymorphic structure, which to a large extent is defined by the anomeric ratio of β to α. Different polymorphs of lactose have different densities, hygroscopicities, melting points, solubilities and speeds of dissolution. Consequently, the crystal form of lactose in dairy powders determines the physical properties of the powder; including flowability, hygroscopicity, solubility and caking.

5.1.2 Current lactose manufacturing processes The manufacture of edible lactose typically commences with the ultrafiltration of whey to produce whey permeate as the feedstock to lactose processing. The whey permeate may be partially demineralised by calcium precipitation and/or nanofiltration. The permeate is then concentrated in a falling film multiple-effect evaporator to 60–70% TS, taking about 6 hours to fill large, specially designed crystallisation tanks. Crystallisation takes place with slow controlled cooling to maximise the size of the lactose crystals, with gentle mixing, taking up to 20 hours. The large lactose crystals are separated in decanter washers, creating a mother liquor by-product containing 20–30% of the lactose and 90% of the ash. The lactose crystals are washed in a refiner washer with cold water (1 : 1 water to lactose ratio). Wash water is often recycled to maximise the yield and minimise waste. The crystals are dried in a fluidised bed drier and milled to the desired size. The yields are in the range 65–75%. The edible lactose process is shown on Fig. 5.3, showing comments on the typical issues associated with each production step. Pharmaceutical grade lactose is manufactured by further refining of edible lactose. This involves redissolving the lactose crystals to 60–65% solids and treating the solution with activated carbon, which adsorbs a number of impurities including riboflavin and peptides (Kellam, 1996). The carbon is removed by flocculation and filtration, and is typically discarded. The concentrated lactose is then crystallised, with recycling to maximise the yield of pharmaceutical grade lactose, see Fig. 5.4. Traditional lactose processes continue to challenge manufacturers despite improvements over the years. Impurities in the lactose-rich whey permeate constrain the efficiency of the process; evaporator fouling by calcium phosphate can reduce operation times to 4–6 hours between cleaning cycles, creating high volumes of cleaning wastes. Purification of lactose

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Whey UF permeate

DCP process pH adjustment, separator washer

Calcium DCP ppte Wash water and lactose losses

Nanofiltration

NF permeate Calcium membrane fouling

Evaporator

Condensate Short runs, waste from CIP cleaning

Crystalliser

Separator

Large tanks, 24hr cycle

Mother liquor 20~30% lactose loss

Water

Decanter washer

Wash water Recycled to evaporator

Drying

Steam, particulates Large yellow crystals

Milling

Potential for amorphous lactose and caking

Packaging

Fig. 5.3

Process chart for the manufacture of edible grade lactose.

is dependant on slow crystal growth and washing procedures. Impurities affect crystal growth and yield, which are aggravated by recycling midstreams, required to improve yields and efficiencies. Further losses occur after crystallisation in the decanter separator, with high wash-water demands and yield loss. The large crystals need to be milled and this can lead to caking and lumping during storage of the milled product (Listiohadi et al., 2005b, c). Entrained impurities and yellow colour reduce the grade and value of the lactose crystals. The yield is 65–75% with best practise. Greater losses can result from poor process control and variable feed quality. Pharmaceutical lactose production requires a second crystallisation, with higher purities achieved by activated carbon and filter aids, which impose further costs and wastage. The mother liquor presents a significant waste disposal problem for lactose manufacturers with BOD5

108

Dairy-derived ingredients Decanted raw lactose from edible process

Hot water, steam

Dissolved 55-60%TS

Activated carbon, filtration agents

Filtration

Filter waste Spent carbon to furnace or waste

Crystalliser

Separator

Mother liquor Recycled to crystalliser or carbon treatment

Cold water

Crystal washer

Wash water Recycled to crystalliser or carbon treatment

Drying

Steam, particulates Large white crystals

Milling

Potential for amorphous lactose and caking

Packaging

Fig. 5.4

Process chart for the manufacture of pharmaceutical grade lactose.

at 160 000 mg/L. If the mother liquor is not quickly used as stockfeed by farmers close to the factory, its disposal can present serious environmental problems.

5.2

Whey and permeate pretreatments

5.2.1 Whey – feedstock for lactose manufacture The feedstock for lactose manufacture is ultrafiltered whey permeate, predominantly from sweet cheese whey. Whey contains 95% of the original water, most of the lactose, 20% of the protein, and traces of fat. There are two main types of whey, sweet whey and acid whey, see Table 5.1. Sweet whey is produced from rennetted cheese, such as mozzarella or cheddar. Sweet whey has higher pH and lower calcium phosphate levels compared with acid whey. Acid whey is produced from lactic acid fermentation for fresh cheese or from hydrochloric acid casein production.

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Table 5.1 Composition of milk and milk permeate; rennetted sweet cheese whey and permeate; lactic whey; hydrochloric acid whey and permeate (Durham, 2000) Component Total solids Lactose Protein Total N NPN Fat Ash Citric acid Sodium Potassium Calcium Magnesium Phosphorus Riboflavin pH

Milk g/100 g g/100 g g/100 g g/100 g g/100 g g/100 g g/100 g g/100 g ppm ppm ppm ppm ppm ppm

12.9 4.8 3.4 0.53 – 3.5 0.75 0.18 410 1300 1100 100 890 1.4 6.8

Milk permeate 5.6 4.7 0.33 0.052 0.032 Ca2+ > Mg2+ >> Ag+ > Rb+ > K+ > NH4+ > Na+ > H+ > Li+ Strong base anion resin: citrate > SCN− > I− > S2O32− > CrO42− > SO42− > HPO42− > NO3− > Cl− > HCO3− > H2PO4− > HCO2− > BrO3− > ClO3− > F− > OH− (Haddad and Jackson, 1990) There are a number of difficulties associated with ion exchange processing; short running times between regeneration, high consumption of regenerant chemicals, salt waste from regenerant chemicals, high water requirements to rinse resins and remove NaOH in particular, plus pH fluctuations when regenerated with strong acids and bases (Jonsson and Arph, 1987). Innovations to ion exchange processing of whey permeate has been reported by Durham (2000) and Durham et al. (1998). This process is based on the ion exchange softening or decalcification of whey permeate. The key to this process is the recovery of monovalent salts from the nanofilter permeate for use as regeneration brine. Moreover, water can also be recovered from the permeate streams for resin rinsing. This work was aimed at addressing the cost and wastage associated with regenerant chemicals, resulting in a cleaner, more cost-efficient softening process. The softened whey permeate has many advantages for lactose processing: nanofiltration with improved flux and no fouling; improved efficiencies for subsequent ion exclusion purification; resulting in longer operation times between cleaning cycles; and minimal fouling during evaporation. Studies on the operating conditions of ion exchange softening have established that calcium removal is greater with treating single strength whey permeate, otherwise high sodium and potassium concentrations in concentrated whey permeate create a competing regeneration effect during ion exchange (Durham et al., 2007). Further studies confirmed that the temperature effect on ion exchange was minimal; therefore operating at

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low temperature was better from a microbiological perspective. It was also observed that protein or peptides can adsorb onto ion exchange resin and negatively impact on ion exchange efficiency. Therefore it is important to monitor ultrafilter membrane integrity to prevent peptides entering the permeate, to maintain process stability for the ion exchange softening of permeates. This softening process has been patented by Durham et al. (1998), covering a four-step process of ion exchange, nanofiltration, recovery of whey brine for the regeneration of the ion exchange column and subsequent purification of lactose by ion exclusion. The process has been licensed by GroupéNovasep. The process was developed as a pretreatment for ion exclusion chromatography for lactose purification; however, it has also been employed as a softening pretreatment to ion exchange demineralisation, with improvement in process and cost efficiencies (Novasep, 2005; Friesland Foods Domo, 2007). 5.2.7 Chromatography purification of lactose The softening pretreatment patented by Durham et al. (1998) creates a softened nanofiltered whey permeate that can be further purified by ion exclusion chromatography to produce a purified lactose stream suitable for direct crystallisation into pharmaceutical grade lactose or conversion into lactose derivatives (Durham et al., 2007). Ion exclusion chromatography is a method whereby sugars (non-ionic components) can be separated from minerals (ionic components) on long, resin-filled columns. In this process, discrete quantities of a sugar/salt mixture such as softened whey permeate, are loaded onto long columns filled with a strong cation resin and eluted with water. The resin is saturated with counter ions in the same composition as the feed, so there is no net ion exchange between the resin and the feed. The ionic material is excluded by the resin and quickly moves through the column (see Fig. 5.5). The nonionic substances partition between the eluent phase and the occluded resin liquid and pass slowly through the column, moving into and out of the resin beads as they traverse the length of the column. The ionic materials emerge first, followed by the non-ionic substances. Ion exclusion separations are improved when the resin is in the monovalent cation form. Monovalent resins have a stronger Donnan potential so that ionic components are more strongly excluded, ensuring that they quickly pass down the column and separate from the non-ionic components. There is no internal ion exchange during elution. Monovalent separations have a single, narrow, mineral peak. The separation of the sugar is also enhanced as the minerals move faster down the column, allowing the resin beads to swell after the minerals pass, more able to absorb the sugar and delay its progress. To achieve monovalent separations, the process relies on pre-treatments such as ion exchange to remove calcium from the feed prior to ion exclusion (Durham et al., 2007).

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Whey permeate containing lactose and minerals

+ + + Lactose

+ +

+

K+ Na+ Cl–

Fig. 5.5 Schematic of ion exclusion resin showing Donnan exclusion of minerals from the charged resin particles and delayed path of lactose permeation through the resin beads (Durham et al., 2004).

There are a number of advantages associated with ion exclusion chromatography. The resin does not require regeneration; therefore no downtime for regeneration, no need for expensive regenerant chemicals, and no accompanying impact of spent regenerant salts on the environment. The elution solvent is water, which can be recovered by recycling the evaporator condensate and reverse osmosis permeate. Anionic and cationic species can be separated in a single pass with up to 99% demineralisation achieved. This process has found widespread application for the recovery of sucrose from beet and cane molasses (Ehrenhuber, 2005; Suhr and Schulze, 2003; Lancrenon, 1997; Chertudi, 1991) and offers similar benefits to lactose processing. Research on chromatographic purification for lactose manufacture can be traced to Harju and Heikkilae (1989), who describe a chromatographic process which recovers lactose from the mother liquor, similar to chromatographic separation of beet sugar molasses being applied commercially in the sugar industry (Harju and Heikkilae, 1989; Harju, 2007). In this process, only the mother liquor is treated by ion exclusion to increase overall lactose yield. More recently, the Ion Exclusion Lactose (IEL) process by Durham et al. (2007) describes a technique whereby the entire lactose stream is recovered by chromatography as purified lactose. In this process, the ion exchanged, softened, nanofiltered whey permeate is separated by ion exclusion chromatography. The mineral peak contains the ionic fraction (potassium, sodium, chloride, non-protein nitrogen, etc.). The lactose peak is essentially pure lactose. The purity, yield and throughput is determined by column length, flowrate and injection size. The resultant purified lactose

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stream is suitable for direct crystallisation into pharmaceutical grade lactose or conversion into derivatives. This process captures the entire lactose stream, achieving an environmental footprint smaller than traditional technology, while avoiding the production of mother liquor. Costings of chromatographic purification of lactose reported by Theobald (2007) show a 2-year return on investment for a plant processing 1 million L UF permeate per day, with a 20% increase yield of lactose, optimal lactose purity, lower water and chemical consumption and lower effluent rejection. 5.2.8 Decolourisation by activated carbon or resin adsorption Whey and permeate solutions also contain coloured compounds, such as riboflavin and Maillard browning products, which are responsible for yellow and brownish colours in crude and edible grade lactose. Riboflavin is sparingly soluble in water, up to 100–250 gµ/ml at room temperature (Association of Vitamin Chemists, 1966). In concentrated permeates, riboflavin tends to co-crystallise with lactose at low temperatures, and is difficult to wash away. Consequently, the final crystallisation temperature can be affected by the amount of riboflavin present in the concentrate. Maillard browning products are formed by reactions between free amino groups of peptides (particularly lysine) and reducing sugars such as lactose. This reaction is favoured by elevated temperatures and long holding times, as occur during evaporation and filling the crystallisation tanks, which can take many hours. Leakage of primary amino groups through the ultrafilter membranes can add to this problem. These coloured compounds can be removed by activated carbon or adsorbent resins. Activated carbon is made from a variety of carbonaceous materials including wood, coal, peat, nut shells, lignite, bone and petroleum residues. The material is dehydrated and carbonised in a kiln in the absence of oxygen, which drives off the volatile matter and produces a rudimentary pore structure. The ‘charcoal’ is then activated by thermal or chemical processes (OMRI, 2002). Activated carbon is employed in the manufacture of pharmaceutical grade lactose. The process involves redissolving (edible grade) lactose crystals and treating the solution with granular or powdered activated carbon to absorb a range of solutes including riboflavin, proteose peptones, Maillard browning products and a variety of proteins. Montilla et al. (2005) found 65–92% colour reduction using 5 mg/L activated carbon. Kellam (1998) reports that extra protein can be absorbed onto the activated carbon by temporarily adjusting the liquor pH. The carbon can be removed from the liquor by flocculation and filtration, and is typically discarded (Kellam, 1998). Activated carbon can be recycled by thermal regeneration, but this is mainly done off-site by specialised companies. Activated carbon has a number of disadvantages, including costly operating expenses, complex thermal regeneration requirements (or waste disposal costs), the risk of microbial growth if conducted at ambient temperatures, and health risks to workers if the carbon is not handled safely (Gula and Paillat, 2005).

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Adsorptive ion exchange resins can also be applied to remove coloured compounds. Ion exchange decolourisation involves a number of phenomena such as ion exchange (mostly anionic interactions regenerated by chloride ions); hydrogen bond interactions between colourant and resin; steric interactions whereby slow flowrates and macroporous resins maximise retention of coloured compounds; or by hydrophobic absorption of nonpolar solutes requiring non-polar regeneration (Gula and Paillat, 2005). Studies by Pragono et al. (2008) on batch columns using macroreticular adsorbent resin, found more than 90% of riboflavin could be removed from a 15% TS lactose solution containing 8–9 mg/L riboflavin without nearing saturation. These studies also demonstrated that sorption followed a pseudo-first-order kinetic model in batch adsorption column system, and that the mechanism for riboflavin adsorption relies on hydrophobic interaction (Pragono et al., 2008). Adsorption resins have been widely applied in sugar processing for decolourisation of beet and cane juice prior to crystallisation (Broadhurst and Rein, 2003; Coco et al., 2008; Garcia Agudo et al., 2002; Gula and Paillat, 2005; Rein et al., 2007; Serpen et al., 2007; Singhal, 2005). However, resin-based decolourisation is less widely reported for whey and permeate processing. Resin based decolourisation has advantages over activated carbon, as the resin can be regenerated and, when combined with membrane recycling of the spent regeneration brine, the waste can be reduced by 90% (Gula and Paillat, 2005), or 75–80% (Coco et al., 2008).

5.3

Lactose crystallisation processes

5.3.1 Fundamentals of lactose crystallisation Industrial crystallisation of lactose is usually performed by slow batch cooling of evaporated permeate, producing large lactose crystals that can be separated, washed and dried. The mother liquor from which lactose is crystallised, comprises a complex mixture of minerals, peptides and organic acids. Consequently, crystallisation functions as a purification and separation process, as much as a crystal recovery step. Crystallisation rates rely on a complex set of interactions determined by lactose solubility, supersaturation, α/β lactose mutarotation, nucleation, temperature, and agitation, and is affected by the type and concentration of impurities in solution (Randolph and Larson, 1988). Lactose crystallisation commences with the formation of nuclei in a super-saturated solution. At high supersaturation, spontaneous nucleation (primary) can occur. Below this region is the intermediate zone, where nucleation will occur only if seed crystals are present, known as secondary nucleation. Next is the metastable zone, where nucleation does not occur, but seed crystals will grow (Lifran, 2007), see Fig. 5.6. Nucleation rates depend on supersaturation, agitation, impurities, seed crystal number and

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Lactose (g / 100g Water)

70 60 50 Labile

40

Intermediate Metastable

Unsaturated

30 20

Lactose solubility Secondary nucleation Primary nucleation

10 0 0

Fig. 5.6

10

20

30 40 Temperature (°C)

50

60

Solubility regions for lactose, showing primary and secondary nucleation thresholds (Lifran, 2007).

size (Kauter, 2003). There are difficulties in accurate measurement of the kinetic parameters for nucleation. Studies by Shi et al. (1990), and Liang et al. (1991) calculated parameters of nucleation kinetics for continuous lactose crystallisation using Mixed Suspension, Mixed Product Removal (MSMPR) population balance technique. However, Butler (1998) found that these studies ignored the large numbers of small crystals below 10 m µ. Butler (1998) subsequently developed an equation representing a closer estimate of nucleation rate, see Equation 5.3. C SNT = e2.992 + 0.0196 T at T < 60°C

[5.3] (Butler, 1998)

The principle driving force for crystallisation is supersaturation. This is achieved by slowly cooling the concentrated lactose solution (65–70% TS) from 70–75°C to 20–10°C. Cooling crystallisation is suited to lactose due to its low solubility (at 22 g/100 g at 25°C, compared with sucrose at 200 g/100 g at 25°C). During lactose crystallisation, the nuclei begin to grow, lactose molecules diffuse towards the crystal, adsorb on the surface and incorporate into the lattice (Hartel and Shastry, 1991). As the crystals grow, α-lactose is depleted and β-lactose mutarotates into α-lactose which continues to be crystallised into α-monohydrate. Concurrently, the solids concentration of the syrup reduces, and cooling is continued maintaining supersaturation and crystal growth. Lactose crystallisation is thus a complex interrelated phenomenon, determined by the kinetics of mutarotation, nucleation and crystal growth, described in a kinetic model by Mimouni (2009).

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Existing practices for cooling crystallisation of lactose take 20–24 hours, including 6 hours to fill the crystallising tank with evaporated whey permeate and 14–18 hours crystallisation. Lactose crystallisation is conducted in specially designed lactose crystallisers, with large cooling surfaces for controlled cooling. Stirring agitator blades ensure that the crystals remain suspended in solution, allowing the crystals to grow but not be damaged by the gentle agitation (Terlet, 2008). Agitation has been found to markedly increase the overall rate of crystallisation by increasing the surface area available for crystallisation from the supersaturated solution (Haase and Nickerson, 1966). Optimum lactose crystals are 200–300 m µ and tomahawk shaped (Ostergaard, 1988). Crystal size distribution (CSD) is one of the main variables governing the performance of downstream processes such as separation, washing and drying. It has been reported that crystals of uniform shape and size, growing under the same environment, do not grow at the same rate and this is known as growth rate dispersion (Hartel, 2001). The resulting CSD affects the end product performance, such as bulk density and flow properties (Vu et al., 2004b). Large crystals with a narrow size distribution, with the least mother liquor inclusion, which can be easily washed, produce the highest quality lactose (Shi et al., 2006).

5.3.2 Effect of impurities on lactose crystallisation The components naturally present in whey and permeate have been extensively studied to determine their effect on crystallisation of lactose (Bhargava and Jelen, 1996; Guu and Zall, 1991; Jelen and Coulter, 1973; Michaels and van Kreveld, 1966; Nickerson and Moore, 1974; Smart, 1988; Smart and Smith, 1991; Visser, 1980, 1984). Some impurities have been found to promote growth, others not to affect it, and yet others to inhibit crystal growth. Impurities influence crystal growth in three different ways: impurities can change the equilibrium solubility, therefore supersaturation; impurities can adsorb onto the crystal faces inhibiting the growth by limiting docking of more growth units; impurities can adsorb on the crystal surface, inhibiting growth, and become included within the crystal lattice, modifying the crystal habit (Mullin, 2001). Bhargava and Jelen (1996) examined the effect of mineral salts on lactose growth, following on from earlier studies by Jelen and Coulter (1973). They confirmed that some salts accelerated growth (LiCl, CaCl2 and MgSO4) while other salts slowed growth (K2HPO4 and KCl). The effect of the different minerals is complex and depends on the species involved. Some minerals increase lactose solubility; for example, calcium increases lactose solubility due to the formation of lactose-CaCl2 complexes (Herrington, 1934). Other mineral impurities may accelerate the surface deposition step of crystallisation, by lowering lactose solubility and accelerating the mutarotation (Jelen and Coulter, 1973). Guu and Zall (1991) found that

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potassium, in particular, decreased lactose growth and yields. Bhargava and Jelen (1996) also found that lactose crystal growth was slowed in the presence of whey permeate, reflecting the presence of potassium in whey permeate. Lactose phosphate has been found to strongly retard lactose crystal growth (Visser, 1988). This has been attributed to the ability of lactose phosphate to dock at the growth sites of all lactose faces, preferentially onto the fastest growing faces 0{ 10}and 1{ 10}. Further studies on the mechanisms of lactose phosphate by Lifran (2007) found that lactose phosphate is incorporated into the crystal since its structure is comparable to lactose and it is anionic at the pH of crystallisation. Consequently, lactose phosphate monovalent anions form a single layer on the crystal surface, creating a negative charge and forming a barrier against further adsorption. The presence of only 60 ppm lactose phosphate was found to be sufficient to cut crystal growth rate by 50% compared to growth rates in ultra pure lactose (Lifran et al., 2006a). Lactose phosphate also decreased the yield, the final median size, and modified the shape of some crystals. Lactose phosphate was found to be concentrated in pharmaceutical grade lactose at levels of 270 to 400 ppm, being concentrated into the lactose during repeated crystallisations. Theories on the origin of lactose phosphate have shown links to cheesemaking processes and the metabolism of lactic acid bacteria, with 10 fold increases in lactose phosphate found in cheese and whey 24 hours after manufacture, compared with the content in fresh milk (Lifran et al., 2006b). Riboflavin has been found to retard the growth of lactose crystals due to its ability to dock onto the crystal growing surface and block growth sites, retarding the growth of that particular face (Michaels and van Kreveld, 1966; Smart and Smith, 1991). However, riboflavin is mainly adsorbed at the surface of the crystals and can be washed off, as opposed to lactose phosphate which is integrated within the crystal structure (Lifran, 2007). β-Lactose has also been found to impact on α-lactose crystal growth. The asymmetric tomahawk shape of α-lactose monohydrate crystals arise from the growth inhibition caused by incorporation of the β isomer in the {011} sector of the crystal (Dincer et al., 1999; Raghavan et al., 2000). Studies have shown that growth under conditions that restrict β-lactose formation result in needle-like crystals (Raghavan et al., 2000). The effect of impurities on lactose quality is magnified when crystallisation is accelerated, as would occur under highly supersaturated conditions or rapid cooling, with the impurities present in the mother liquor entrained in the lactose crystals. Smart and Smith (1991) found calcium phosphate and calcium sulphate salts co-precipitated with the lactose when their solubility had been exceeded during crystallisation. Impurity analysis of lactose crystals by Butler (1998) also revealed entrapped calcium phosphate and adsorbed riboflavin. Consequently, rapid crystallisation is not desirable when it results in the incorporation of large amounts of impurities within the crystal.

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5.3.3 Innovations in lactose crystallisation Lactose is generally crystallised in cooling batch crystallisers in a highly empirical process which can be unpredictable, with difficulties in optimising for quality, recovery, efficiency, capital cost and energy consumption (Shi et al., 2006). The control of lactose crystallisation has been the subject of much recent research, with a range of crystallisation parameters being the focus for improvement, including; cooling profile (Shi et al., 2006), seeding (Guu and Zall, 1991; Jayaprakasha et al., 1994; Vu et al., 2004a), sonocrystallisation (Bund and Pandit, 2007a; Vu et al., 2003), continuous crystallisation (Henningfield and Dinesen, 2003); recycling mother liquor (Cloidt and Lehmann, 2007), crystallisation from solvents and ternary systems (Bouchard et al., 2008; Brito and Giulietti, 2007; Bund and Pandit, 2007b; Lifran et al., 2005) and permeate crystalliser driers. A process employing a controlled cooling profile to achieve lactose crystals with uniform size and narrow size distribution has been patented by Shi et al. (2006). This patent recommends that the concentrated lactose permeate be initially rapidly cooled from 80°C to 50°C and then held at 50°C to encourage nucleation, thereafter gradually cooled to 30°C to assist crystal growth and maximise yield. The resulting crystals with narrow size distribution can be more efficiently washed and separated. This process relies on primary nucleation as the source of seeds and control of the cooling profile to manage the size distribution of the resulting crystals. Seeding is rarely employed for control of industrial crystallisation of edible lactose, yet there has been consistent research demonstrating the value of seeding for improved yield and control over crystal size distribution. Ideally, seeded crystallisation is conducted within the metastable solubility zone; that is where seed crystals grow but nucleation does not occur. This allows for better control of the crystallisation process, with the crystal size distribution controlled by seeding and controlled cooling (Lifran, 2007). Guu and Zall (1991) and Jayaprakasha et al. (1994) both showed that seeding increased the relative gross yield. Vu et al. (2004a) found that seeding could speed up crystallisation growth rate with more consistent product quality, achieving high yields of crystals with narrow size distributions compared to self-nucleated batch crystallisation. A dynamic optimization model developed by Vu et al. (2006) has been used to investigate and compare seeded and non-seeded batch cooling, semi-batch and continuous cooling crystallisation of lactose, to identify conditions for producing uniform size distribution at the highest yield. Semi-batch seeded crystallisation was found to achieve higher yields in shorter times, compared with batch cooling. Semi-batch mode involves cooling in accordance with a defined temperature profile, while syrup is continually fed into the tank until it is filled. Continuous feeding of fresh syrup maintains the high driving force for crystallisation and constrains temperature drops (Vu et al., 2006). These results also suggest that

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evaporative batch operation is faster than batch cooling; however, its implementation is more complicated and nucleation is difficult to control. A continuous evaporative crystallisation apparatus for lactose has been patented by Henningfield and Dinesen (2003), whereby the concentrated permeate or lactose is simultaneously concentrated and crystallised whilst being conveyed along a horizontal evaporative crystalliser with a hollow screw shaft for agitation and cooling. The resulting lactose crystals are recovered from the crystalline slurry (at 75% TS). Another recent patent in lactose crystallisation describes a process whereby a part of the desugarised permeate (mother liquor) is fed back (recycled) to the lactose concentrate prior to the evaporation step and reduces yield loss from the mother liquor. The process includes demineralisation of the permeate by nanofiltration, electrodialysis or ion exchange to maintain purity to the crystalliser (Cloidt and Lehmann, 2007). Lactose crystals with uniform size distribution have been produced using drop-based microfluidic crystallisers, whereby supersaturated solutions of lactose are expelled dropwise from narrow tubing at 100 m µ , 150 µ m or 300 µ m I.D., creating crystals in the size range close to that of the tubing diameter. Dombrowkski et al. (2007) report CSD at 16% compared to 40% for bulk crystallisation, while drops containing single crystals had 7% CSD. Sonocrystallisation has been investigated to initiate nucleation and accelerate the growth of lactose crystals (Bund and Pandit, 2007a; Vu et al., 2003, 2004b). Sonocrystallisation thus provided a solution to the difficulties associated with long induction times associated with natural nucleation of lactose (Raghavan et al., 2001). Studies have shown that ultrasound enhances nucleation, with higher ultrasound levels resulting in higher crystal yields and smaller size particles, due to the high number of nuclei that have been generated, and also that agglomerates are broken-up (Vu et al., 2004b). Sonocrystallisation has also been combined with solvent addition and cooling to produce fine seed crystals for use in controlling bulk crystallisation of lactose (Vu et al., 2003, 2004b). The use of solvents for accelerated crystallisation to control crystal shape and size distribution, has also been the subject of research in a number of studies over the years (Olano et al., 1977; Singh et al., 1991; Lifran et al., 2002, 2004b; Bund and Pandit, 2007a). Bund and Pandit (2007a) found that alcoholic crystallisation increased the yield of lactose from concentrated deproteinated paneer whey (containing 19.5% w/v lactose) up to 90% with 65–85% ethanol at 7°C. The method described by Lifran et al. (2002) involves staged alcohol (preferably ethanol) addition for demineralisation and then controlled crystallisation, producing free-flowing pharmaceutical grade, rosette shaped crystals with unique properties. Work by Vu et al. (2004b) found that solvent addition and sonocrystallisation could be used to produce very fine crystals for pharmaceutical applications or seed lactose, thus avoiding the problems associated with amorphous lactose created during size reduction milling (hammer, ball or jet mill).

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Finally, recent literature describes a number of permeate crystalliser driers used for the production of free-flowing dried permeate. These processes employ special evaporators capable of making permeate pastes at very high concentrations (78–86% TS) yet avoiding heat damage. Evaporation is followed by rapid crystallisation to create very fine lactose crystals which are dried in specially constructed driers to produce a free flowing powder. The Niro Thixotherm process is named after the thixotropic nature of highly concentrated crystallised lactose. The permeate is dried in a RosinaireTM paddle dryer with a screw conveyor to cure and crystallise the permeate followed by drying and cooling in a combined back-mix/plug-flow fluid bed drier (Pisecky, 2005). The Anhydro Paraflash process utilises a Paraflash forced circulation flash evaporator to concentrate the permeate to 78% solids, accompanied by continuous crystallisation of lactose and drying in a spin flash dryer (Andersen, 2005). The Relco Keller drying system concentrates the permeate to 78% solids, followed by triple C (cooling concentrator crystalliser), then an air lift drier, an integrated two-stage fluid bed (Keller, 2007). Carlisle Process Systems also developed new approaches for drying acid whey and permeates which are prone to stickiness (Boersen and Veldmeijer, 2003), described by Neville (2006), in a modern, purpose-built whey and permeate drying plant in the Netherlands.

5.4

Post handling – separation, washing and drying crystals

5.4.1 Decanting and washing crystals The crystalline lactose slurry exiting the crystalliser requires separation and washing to remove the mother liquor and prepare for drying. There are a number of different types of separators available; solid bowl decanter (horizontal shaft centrifugal decanter), screen bowl decanter (worm/screen centrifuge) (Brinkman, 1976; Gesterkamp, 1994; Mans, 1994; Bylund, 2003; TEMA, 2007), and hydrocyclones (Zeevalkink et al., 2005). Decanter separators are deployed in series, with the crystal slurry first being centrifuged in the solid bowl decanter to separate the mother liquor and raw lactose. Efficient operation of the decanter centrifuge is facilitated by large crystals (200–300 µ m) with narrow size distribution, ensuring a free-fl owing crystal slurry, from which the mother liquor can be drained and separated. Too many fines will clog the separator and large amounts of mother liquor will be retained, with the resulting crystals containing higher levels of impurities and higher levels of moisture impacting on drier running costs. The raw lactose is then washed and centrifuged in a series of screen bowl centrifuges. The quantity of wash water required to wash the crystals depends on the CSD, the number of wash steps and the counter current recycling of wash water (Mans, 1994). Wash water corresponding to 10% of the feed quantity has been reported by Ostergaard (1988) and van den

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Bos (1987), while Bylund (2003) report 1 : 1 wash water ratio with the lactose processed. The wash water can be recycled back to an earlier step in the process to recover lactose and minimise losses from washing. The purity of the wash water impacts on the purity of the lactose; thus, higher grades of lactose require demineralised water or RO permeate, while potable water would suffice for edible grades of lactose. After decantation, the lactose is 88–90% TS and requires drying. 5.4.2 Drying lactose crystals Moist lactose crystals exiting decanter separators are dried in a fluidised bed drier to produce a free-flowing powder with a final moisture content of 4.5–5.5% (in α-lactose monohydrate). This applies to both edible grade lactose and pharmaceutical grade lactose that has been further processed by secondary purification and crystallisation. The fluidised bed drier is limited by a product temperature of 93°C, otherwise β-lactose will be formed from the residual amorphous lactose. Above 100°C, further moisture will be driven out, forming anhydrous unstable α-lactose (Brinkman, 1976). The fines from the drier exhaust should be collected in a bag filter to maintain air quality in the surrounding area (GEA, 2008b). Drying time impacts on product quality: if the lactose is dried too fast then amorphous lactose on the surface of the crystals dries as amorphous glass and increases the risk of caking during storage. The amorphous lactose should be given sufficient time to crystallise as α-lactose monohydrate during drying. The air used to dry the crystals should be dehumidified and monitored for RH% (GEA, 2008b), to prevent caking problems during storage. The lactose should also be cooled prior to packing, otherwise when placed in a cool store, moisture will migrate from the hot region within the centre of the bag to the cool region at the surface; and if there is enough moisture present in the powder, capillary condensation between particles will cause liquid bridging and dissolution, which eventually leads to caking and lumping as the moisture is redistributed when the temperature has equilibrated (Paterson and Bronlund, 2009). 5.4.3 Grinding and milling Lactose crystals are normally milled and sieved to a range of size specifications. There is a range of lactose sizes available on the market: for example, 60 Mesh (40–65% 250 m µ ), 100 Mesh (60–80% 100 m µ ), 200 Mesh (75–85% 75 µ m), 350 mesh (>65% 45 m µ ), achieved by milling and sieving. Milling has the potential to cause changes in the crystalline lactose. Otsuka et al. (1991) found that the crystal water of α-monohydrate was converted into absorbed water by mechanical stress, and that isomerisation of lactose occured in the solid state during grinding. Crystallinity decreases with grinding time, resulting in the formation of amorphous lactose with the

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release of the water of crystallinity (Otsuka et al., 1991). Amorphous lactose is very susceptible to elevated humidity levels, with lumps of black mould found to occur within the sacks of lactose when there have been insufficient control over humidity during drying and milling. Furthermore, there is also an increased propensity to lump and cake as the amorphous lactose recrystallises into α-lactose monohydrate during storage (Listiohadi et al., 2005a).

5.5

Specialised pharmaceutical products

5.5.1 Spray-dried lactose Spray-dried lactose consists of spherical particles of small α-lactose monohydrate crystals, glued together in an amorphous lactose matrix (Vanderbist et al., 1998). It is commonly used as an excipient for direct compression tableting, filler and flowing agent in pharmaceutical preparations (Patel and Chen, 2008). Its spherical shape is advantageous for use in high-speed tableting machines used by pharmaceutical manufacturers. Spray-dried lactose is manufactured from redissolved pharmaceutical grade lactose, which has been pre-crystallised by flash cooling and rapid mixing in specialised tanks to encourage a shower of very fine crystals (Jensen, 1987). The lactose slurry is atomised in the spray drier and the semi-dried particles are subsequently processed in vibratory fluidised bed driers to encourage secondary crystallisation into α-lactose monohydrate. An agglomerated powder can be produced by recycling the fines into the drying chamber (Patel and Chen, 2008). Agglomerated powders have improved flowability, reduced dustiness and instant properties. Pre-crystallisation has a number of advantages in that it is possible to process concentrates up to 60% solids with improved process efficiency if the lactose has been subject to crystallisation. It is not possible to spray dry non-crystallised concentrate over 42–45% due to risk of blockages (Jensen, 1987). Pre-crystallisation into α-lactose monohydrate also creates a product that is less hygroscopic and more stable, with improved flowability and drug dispersion properties (Schuck and Dolivet, 2002). Spray-dried agglomerated lactose typically contains 9–12% β-lactose, present in an amorphous matrix which is usually 15–20 wt.% of the total product (Bolhuis et al., 2004). Controlling the degree of crystallisation in spray-dried lactose is the key factor for controlling many of its properties, including density and porosity (Trivedi and Axe, 2001), solubility (Blagden et al., 2007; Pokharkar et al., 2006), flowability (Fitzpatrick et al., 2007) and sorption characteristics (Chiou and Langrish, 2008; Lehto et al., 2006). The degree of pre-crystallisation, the inlet air temperature and the particle temperatures throughout the dryer have been established to be important in determining the final crystallinity of the product, storage stability and propensity to cake (Chiou and Langrish, 2008).

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5.5.2 Anhydrous lactose Anhydrous lactose, containing high levels of crystalline β-lactose, is used as a pharmaceutical excipient for wet granulation and direct compression, and for Direct Powder Inhalation (DPI) formulations. β-Lactose is more soluble than α-lactose monohydrate and has low water content, which is advantageous if the pharmaceutical active is sensitive to moisture (Vanderbist et al., 1998). Roller-dried anhydrous lactose also yields more reproducible results as a carrier for DPI, due to its highly crystalline, anhydrous nature (Chow et al., 2007). Anhydrous lactose is manufactured by redissolving pharmaceutical grade lactose in purified water then roller-drying between two heated counter-rotating drums. The metal rollers are heated from inside with condensing steam (120–160°C) (Shilton, 2002). The product to be dried is spread as a thin film onto the surface of the hot drum and after one revolution is scraped off by knives (Vanderbist et al., 1998). Since the lactose is dried at ~15 g · 100 g−1 protein; similarly, calcium caseinate is readily dispersable but not as soluble as sodium caseinate. Rennet casein, despite its insolubility, is the preferred protein type for use in block processed cheese or analogue/substitute pizza cheese. In both products, it behaves like fresh natural rennet-curd cheese, conferring a high degree of elasticity and firmness to the unheated processed cheese, and moderate meltability. Partial replacement of young Cheddar cheese (intact casein content, ~93 g · 100 g−1 protein) with commercial rennet casein (intact casein, ~99 g · 100 g−1 protein) results in firmer processed cheese that melts to a lower extent on heating. This effect may be attributed to the higher calcium phosphate content of rennet casein, ~33 mg·g−1 casein compared to Cheddar cheese (~27 mg·g−1 casein). Rennet casein also contributes to the desired degrees of stringiness and stretchability in the hot processed mass during the manufacture of analogue pizza cheese, an attribute that is highly desired by the manufacturer as it gives the appearance of natural low moisture Mozzarella cheese to the latter. However, despite this, commercial analogue pizza cheeses made from rennet casein generally possess inferior

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stringiness compared to natural Mozzarella cheese when subsequently melted (Guinee et al., 2000a). This probably reflects differences in the degree of protein hydration and microstructure. Recent work in the author’s laboratory has shown the level of protein hydration (as measured by the degree of water extractable protein when using a 2 : 1 water : cheese slurry) in analogue cheese made with a very low level of disodium orthophosphate (~0.75 g · 100 g−1) was much higher than that in freshly-made Mozzarella cheese (~40 versus ~5 g · 100 g−1); the level of water-soluble protein in PCPs and ACPs is generally much higher (~60–80 g · 100 g−1) owing to the higher level of emulsifying salts used (e.g. 1–2 g · 100 g−1). Moreover, microstructural studies have shown that the aligned calcium phosphate paracasein fibres (with interspersed fat pools/columns showing similar orientation) which give natural molten Mozzarella its stringiness are absent in analogue pizza cheese, which consists of discrete rounded fat droplets of varying size in a continuous protein phase (Guinee et al., 2000a; Kindstedt et al., 2004). Caseinates (especially sodium) find most application in processed cheese spreads (PCSs), where their high water-binding capacity and good emulsifying properties promote a desired creaming effect. Gouda et al. (1985) reported that full replacement of non-fat cheese solids by calcium caseinate caused deterioration in spreadability of Cheddar PCS, probably due an excessive creaming effect. However, partial replacement in a formulation (with skim milk powder, calcium caseinate, ripe Cheddar, butter oil and emulsifying salt at respective levels of 6–8, 5–7, 15, 14, and 3%, w/w) improved the meltability of the PCS, suggesting a desirable level of creaming. Caseinates may be used in spreadable imitation cheese products (Marshall, 1990; Hokes et al., 1989). Acid casein is generally not used in processed cheese manufacture because of its insolubility, low pH, and its depressing effect on the pH of the processed cheese blend, which in turn reduces the degree of dissociation of the emulsifying salts and their ability to sequester calcium from other proteins (e.g. cheese protein, or rennet casein). Consequently, the use of acid casein at quantities of 1 to 3% (w/w) of the processed cheese formulation can markedly extend the product make time, unless the pH of the blend is increased to its normal value (e.g. ~5.8 to 6.0) via addition of alkali and/or an emulsifying salt blend with the desired pH-buffering effect. A comparison of the functionality of acid casein and rennet casein on the melt properties of model processed cheeses (made from the casein powder, vegetable fat, water and emulsifying salts) was made by Savello et al. (1989). The response of meltability to casein type depended on the type of emulsifying salt and the pH to which the acid casein was adjusted (upwards) during processing. Acid casein (pH adjusted to ~7.0 to 7.7) gave model processed cheeses with higher meltability than those made with rennet casein, using disodium hydrogen orthophosphate or tetrasodium pyrophosphate, and an opposite effect when using sodium aluminium phosphate or trisodium citrate.

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Casein hydrolysates may be used successfully as a means of partially replacing sodium orthophosphates in processed cheese manufacture (Kwak et al., 2002), despite the fact that the pH of the processed cheeses containing the hydrolysate is significantly lower than the control (~5.7–5.9 compared with 6.3). This was attributed to the increased emulsifying capacity of the hydrolysate over the parent protein, sodium caseinate. However, when added at a level of 3%, w/w, to replace the emulsifying salt completely, the hydrolysate gave a PCP that on cooking had a high flowability but excessive oiling-off. Whey proteins In practice, whey proteins are added to processed cheese formulations mostly in the form of whey powder (e.g. sweet whey powder with ~12–15 g · 100 g−1 protein), which is used as a cost-effective filler to impart a mild sweetish taste and a smooth consistency, especially desirable in highmoisture processed cheese spreads and dips. Whey protein concentrates (WPCs) or whey protein isolates (WPIs), though used less frequently, are included in formulations for specific functions such as (i) to give low-flow or flow-resistance in ‘controlled–melt’ PCPs targeted to specific cooking applications (e.g. cheese insets/pieces in meat-based products, fried cheese), and (ii) to confer ‘body’ (stiffness and viscosity) to high moisture spreadable PCPs. Numerous studies have investigated the effects of added whey proteins on the texture and cooking characteristics of PCPs (Schulz, 1976; Savello et al., 1989; Hill and Smith, 1992; Gupta and Reuter, 1993; Kaminarides and Stachtiaris, 2000; Gigante et al., 2001; Mleko and Foegeding, 2000; 2001; French et al., 2002; Mleko and Lucey, 2003; Mounsey et al., 2007). These studies have generally shown that the use of whey proteins, even at relatively low levels (1–2 g · 100 g−1), as a substitute for cheese protein or casein at similar levels, reduces the flowability of the heated PCP to an extent that increases as the level of substitution increases (Savello et al., 1989; Gupta and Reuter, 1993; Gigante et al., 2001; Mleko and Foegeding, 2001; Mounsey et al., 2007). Indeed, an early patent (Schulz, 1976) describes a process for the manufacture of PCP that is resistant to flow on cooking, based on the addition of a heat-coagulable protein (3–7%, w/w, lactalbumin, egg albumen), at a temperature 3 g · 100 g−1) significantly reduced the flowability of the PC, especially when a high proportion of young cheese was used. The depressing effect of CWPt on meltability of PCPs may be associated with a number of factors including inter alia: • a higher emulsion stability (Thomas, 1970) and/or degree of emulsification (Gupta and Reuter, 1993); • the formation of large whey–protein casein aggregates in the CWPt (O’Kennedy and Mounsey, 2009) which, on inclusion in processed

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cheese, impede slippage of the protein layers and thereby restrict heatinduced flow, similar to the melt-inhibitory effect observed by McMahon et al. (1996) in low-fat Mozzarella cheese made from milk with added Novogel (a proprietary blend of microcrystalline cellulose and guar gum); and/or • the formation of a non-melting whey protein network (Mleko and Lucey, 2003). Co-precipitates are not widely used in the food industry owing to their generally poor functionality (low solubility, emulsification) as an ingredient, and adverse effects on the meltability/flowability of processed cheese products. However, new approaches aimed at controlling heat-induced denaturation and aggregation of whey protein, and the size/gelation capacity of the resultant reaction products may improve the functionality of CWPts (Mleko and Foegeding, 1999; Donato and Guyomarc’h, 2009). Mounsey et al. (2007) investigated the effect of varying the pH of skim milk to values greater (7.5, 9.5) or lower (3.5) than the control (~6.6), prior to heating and re-acidification to pH 4.6, on the functionality of the resultant CWPts in model processed cheese products. The CWPts were added at 8 g · 100 g−1 as a substitute for acid casein powder and the level of whey protein (g · 100 g−1) in the processed cheese varied from 1.37 (for pH 7.5 and 9.5) to 0.96 (pH 3.0) due to the effect of pH at heat treatment on whey protein denaturation and recovery. Increasing the pH at heating yielded CWPts that markedly improved the meltability, which, for the processed cheese made from the pH 9.5, CWPt was similar to the control processed cheese containing 8 g · 100 g−1 acid casein. Conversely, reducing the pH had the opposite effect. Micellar Caseins (MC) and Milk Protein Concentrates (MPCs) Micellar caseins are produced from skim milk, which is subjected to microfiltration and diafiltration to remove whey proteins, lactose and soluble salts (Kelly et al., 2000). MPCs are similarly manufactured except that the skim milk is ultrafiltered instead of microfiltered so as to recover the whey protein along with the micellar casein (Guinee et al., 2006). For both ingredients the diafiltered retentates are spray dried to yield powders with protein levels of ~≤82 g · 100 g−1, being comprised of casein micelles and colloidal ash in MCs and micellar casein, whey proteins and ash in MPCs. The use of MC or MPC at a level of 6.3 g · 100 g−1, as a substitute for young Cheddar cheese protein (intact casein ~90 g · 100 g−1 protein) in the formulation reduced flowability of the melted processed cheese, with the effect being more pronounced for the MPC (Fig. 20.5). The lower flow with the MPC is most likely due to heat-induced aggregation and/or gelation of whey proteins, present at a level of ~1.3 g · 100 g−1 in the processed cheese, even though other factors (such as the higher intact casein content of the added MPC) may also contribute.

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(a) 100

Firmness (N)

80 60 40 20 0 Control

PC1

MPC1

Control

MC1

Control

MC

MPC2

(b) Flow (Schreiber Test) (%)

70 60 50 40 30 20 10 0 MPC1

MPC2

MPC1 Ingredient

MPC2

(c) Flow (Price-Olson Test) (%)

600

400

200

0

Fig. 20.5 Effect of partial replacement of Cheddar cheese with different ingredients (MC and MPC) on the physical properties processed cheese. The ingredients were added to replace 25% of the protein content of the Cheddar cheese. To ensure similar levels (g · 100 g−1) of moisture (~47), fat (~25) and protein (20.5) in all processed cheeses, anhydrous butter oil and water were added along with the ingredients. Treatment processed were made using Cheddar cheese only (control), and using Cheddar cheese plus micellar casein powder (MC), milk protein concentrate powder 1 (MPC1), or milk protein concentrate powder 2 (MPC2). All powders had 80 g · 100 g−1 protein, and the MPC powders differed with respect to heat treatment applied during manufacture (low heat, MPC1; high heat, MPC 2). Data are the means of three replicate trials, and error bars show standard deviations.

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Dairy-derived ingredients

20.4.3 Cheese Base (CB) and Ultrafiltered Milk Retentate (UFMR) Production of CB generally involves ultrafiltration and difiltration of skim milk (typically to 20–25 g · 100 g−1 dry matter), inoculation of the retentate with lactic culture, incubation to a set pH (5.2–5.8), pasteurisation and scraped-surface evaporation to ~60 g · 100 g−1 dry matter (Ernstrom et al., l980; Sutherland, 1991; Ganguli, 1991). However, rennet may be added to the retentate to form a curd from which a small quantity of whey is removed (compared to that in natural cheese manufacture) and which is dry-salted salted and pressed, and stored as natural cheese. The retentate may also be treated with lipase to enhance the flavour of the final PC; it was claimed that up to 80%, w/w, of Ras cheese solids could be replaced by the lipasetreated retentate, with the resultant PCs having flavour and consistency considered to be superior to those of the control (Aly et al., 1995). Hyde et al. (2002) describes the preparation of cheese base by the acidification and cooling of a blend comprising one or more powdered milk protein ingredients, milk fat, NaCl, food-grade acid and/or preservative. Increasing the level of substitution of natural cheese by CB, made in the conventional manner, or UFMR, normally results in a ‘longer-bodied,’ firmer PCP which is less flowable on heating (Collinge and Ernstrom, 1988; Tamime et al., 1990; Younis et al., 1991). The lower flowability may be attributed to a number of factors including: • a higher degree of intact casein in the cheese base, • the presence of whey proteins in the cheese base (~8.7%, w/w), which are denatured and complex with para-κ-casein to form a pseudo-gel at the high processing temperature (85–90°C for 3 min) (cf. Doi et al., 1983a,b, 1985). The tendency of whey proteins to aggregate and gel is probably accentuated by the high levels of protein and soluble calcium in the cheese (Doi et al., 1983a,b; O’Kennedy and Mounsey, 2009; Donato and Guyomarc’h, 2009). However, the effects on flowability vary depending on the method of preparation of the CB and UFMR, and the subsequent heat treatment during processing: Reducing the pH of milk, from 6.6 to ≥5.2, prior to UF resulted in CBs with lower calcium levels and PCPs with improved meltability (Anis and Ernstrom, 1984); (ii) Rennet treatment of the UF retentate results in poorer meltability (Anis and Ernstrom, 1984), an effect which may be attributed to the higher degree of interaction between β-lactoglobulin and para-κcasein (than with native casein) during subsequent processing (Doi et al., 1983 a,b); (iii) Treatment of retentate with exogenous proteinases (e.g. Savorase-A, and enzymes from Aspergillus oryzae and Candida cylindracea), which increase the level of proteolysis in the CB, yields PCPs that are (i)

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softer and more meltable than those made with untreated CB (Sood and Kosikowski, 1979; Tamime et al., 1990, 1991); Increasing the processing temperature in the range 66 to 82°C results in PCPs with reduced meltability, an effect attributed to heatdenaturation and gelation of whey proteins at the higher temperatures, especially when rennet-treated CB is used (Collinge and Ernstrom, 1988).

20.4.4 Other dairy ingredients Skim milk powder Addition of skim milk powder to PCP blends at a level of 3–5%, w/w, results in softer, more spreadable products (Kairyukshtene and Zakhrova, 1982). However, higher levels (7–10%, w/w) lead to textural defects such as crumbliness and lack of cohesiveness (Thomas and Hyde, 1972; Kairyukshtene and Zakhrova, 1982) and the powder may remain undissolved. However, a high level may be added if the skim-milk powder is first reconstituted and then precipitated by proteolytic enzymes or citric acid, and the curd added to the blend (Thomas, 1970). Lactose Added lactose, in the range of 0 to 5%, w/w, resulted in lower spreadability, lower water activity and increased propensity to non-enzymatic browning in PCPs during processing (especially at a high temperature) and storage (Piergiovanni et al., 1989; Kombila-Moundounga and Lacroix, 1991). Hong (1990) found that replacement of experimental cheeses by lactose at levels of 5 to 20%, w/w, reduced the firmness of PCP. Excess lactose may also increase the propensity to crystallisation in PCPs during storage, with the formation of mixed crystals containing various species, e.g. Ca, P, Mg, Na, tyrosine and/or citrate. Owing to the relatively high level of bound water in PCPs (a maximum of 1.6 g/g solids-non-fat; Csøk, 1982), the effective lactose concentration in the free moisture phase may easily exceed its solubility limit (~15 g/100 g H2O at 21°C). This may result in the formation of lactose crystals, which could serve as nuclei for crystallisation of mineral species that are supersaturated (Guinee et al., 2004).

20.5

Conclusions

Processed cheese products (PCPs) are composites formulated from natural cheese, water, emulsifying salts and optional ingredients, the type and level of which depend on the category of the PCP, e.g. named variety processed cheese, processed cheese or processed cheese preparation. The formulation is blended and heated to ~75–85°C while shearing continuously to give a hot uniform molten PCP which is hot-packed and stored chilled. While

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natural cheese is the major ingredient, accounting for ≥51 g · 100 g−1 in all categories of PCPs, dairy ingredients can be added at substantial levels (e.g. 10 g · 100 g−1) to some categories such as processed cheese preparations, as defined by the Codex Alimentarius Commission (FAO/WHO, 2008). Calcium phosphate paracasein is the main protein in rennet curd cheeses (e.g. Cheddar, Gouda), which are the main varieties used in PCPs. This protein is inherently insoluble because of intermolecular calcium and/or calcium phosphate mediated linkages. Hence, heating cheese to a high temperature (e.g. 90°C) while shearing, is conductive to protein aggregation, destabilisation of the fat globule membrane, fat coalescence, and leakage of free fat and moisture. However, the addition of emulsifying salts, such as sodium orthophosphate and/or citrate, prevents such an occurrence and promotes the formation of stable uniform PCPs. The salts do this by increasing the pH of the blend (e.g. ~5.7 to 6.0, compared with 35°C, the fat is liquid, but upon cooling and storage, the dispersed fat droplets will be solid. Butter and margarine are examples of liquid water droplets in a solid fat phase; these are special cases in terms of emulsified dairy products and will not be discussed in this chapter.

21.1.2 Microemulsions and macroemulsions Emulsions can be divided into two main classes based on their thermodynamic stability. Microemulsions are thermodynamically stable, and macroemulsions are metastable. Macro-emulsions (commonly referred to simply as ‘emulsions’), as stated previously, are dispersions of droplets in an immiscible fluid. Breaking up the dispersed phase into droplets requires energy to overcome the surface tension forces between the two phases, and hence they are thermodynamically unstable and will eventually collapse back into their component phases. The droplet diameter is generally in the range 0.1–10 µm. Microemulsions overcome the thermodynamic barrier by reducing the interfacial tension to near-zero. This is achieved by high concentrations of surfactants (and often co-surfactants, mixtures of oils and oil soluble compounds). The result is that when the two phases come into contact, they spontaneously emulsify to form a microemulsion with very small droplet diameters (70–75°C, whereas BSA and Igs begin to denature at ~65°C. The order of sensitivity of various whey proteins to heat-induced denaturation has been reported to be: Igs > BSA > β-LG A > β-LG B > α-LA (Dannenberg and Kessler, 1988; Oldfield et al., 1998a). The kinetics of denaturation of the whey proteins are quite complex, with the reaction characteristics showing marked changes above 80–100°C. The denaturation reaction shows a non-linear Arrhenius plot; there is a marked change in temperature dependence at about 80–90°C for both α-LA and β-LG. The apparent activation energies are in the range 260–280 kJ/mol for β-LG and 270–280 kJ/mol for α-LA at temperatures below 90°C (Dannenberg and Kessler, 1988; Oldfield et al., 1998a). At higher temperatures, the activation energy is lower, ranging from 54 to 60 kJ/mol for β-LG and from 55 to 70 kJ/mol for α-LA, indicating chemical (aggregation) reactions.

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In the milk powder industry, the extent of denaturation of the whey proteins is expressed as the whey powder nitrogen index (WPNI). SMP is dissolved in water and then saturated with NaCl solution, followed by filtration, which retains denatured whey proteins and casein. The filtrate is analyzed for its protein content and the value is expressed as the WPNI, which is the quantity of undenatured whey proteins per gram of powder. Thus, as the extent of denaturation of the whey proteins increases, the WPNI decreases. On the basis of the WPNI, SMPs can be classified into low, medium and high heat types. Following denaturation, the whey proteins can interact with the casein micelles, more specifically with κ-casein in the micelles. Two disulphide bridges and a free thiol group in the native structure of β-LG play an important role in these interactions. The interaction between β-LG and κ-casein has been shown to involve the formation of disulphide bonds. Heat treatment causes native β-LG to interchange into non-native monomer and non-native disulphide-bonded dimer, which then interact with κ-casein. β-LG can also form complexes with αs2-casein and α-LA (Patel et al., 2006). Through their interactions with micellar κ-casein at the micelle surface, some of the denatured whey proteins become associated with the casein micelles. Some remain in the serum, where they may form aggregates with other whey proteins or with serum κ-casein. When the temperature of milk is increased gradually above 70°C, as in an indirect heating system, most of the β-LG and most of the α-LA associate with the micelles, whereas, when milk is heated rapidly, as in a direct heating system (e.g. direct steam injection), only about half of the β-LG and half of the α-LA associate with the micelles (Corredig and Dalgleish, 1996; Oldfield et al., 1998b). Oldfield et al. (1998b) suggested that there are at least three possible species of denatured β-LG that could associate with the micelles: (i) unfolded monomeric β-LG, (ii) self-aggregated β-LG and (iii) β-LG/α-LA aggregates. The relative rates of association of these species with the casein micelles depend on temperature and heating rate, which in turn affect the relative rates of unfolding and the formation of the various aggregated species. At high temperatures and fast heating rates, all whey proteins begin to unfold in a short period of time, thus presenting more opportunity for unfolded monomeric β-LG to self-aggregate, which consequently is likely to associate with the casein micelles less efficiently. These β-LG aggregates could protrude from the micelle surface, providing steric effects for further β-LG association. In addition, these aggregates may have their reactive sulphydryl group buried within the interior of the aggregate, and therefore unavailable for sulphydryl–disulphide interchange reactions with micellar κ-casein. The formation of unfolded β-LG may be promoted by long heating times at low temperatures, or by heating at a slow rate to the required temperature. These unfolded monomeric β-LG molecules would be expected to penetrate the κ-casein hairy layer with greater ease and have a readily accessible sulphydryl group.

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In addition to time and temperature of heating, several other factors influence the extent of association of denatured whey protein with the casein micelles; these include the pH of the milk prior to heating, levels of soluble calcium and phosphate, and the milk solids concentration (Singh, 1995). Heating at pH values less than 6.7 results in a greater quantity of denatured whey proteins associating with the casein micelles, whereas at higher pH values, whey protein κ-casein complexes dissociate from the micelle surface, apparently because of dissociation of κ-casein (Singh and Fox, 1985). The interactions of whey proteins with the casein micelles causes changes in the casein micelle size during heating at temperatures up to 100°C, but the change in micelle size is dependent on the pH of the milk at heating, which also influences the extent of association of denatured whey proteins with the micelles. At pH 6.5, where about 70% of the denatured whey proteins are associated with the micelles, the average micelle size increases by about 35 nm. At pH 6.7, where the level of whey protein association is about 30%, the micelle size increases by only about 5 nm (Anema and Li, 2003). Association of whey proteins modifies the surface of the casein micelles and is likely to influence the functionality of the reconstituted milk powders, particularly rennet clotting behaviour, solubility and heat stability.

25.3.2 Evaporation Falling film evaporators are predominant in the dairy industry and are designed to remove as much water from the milk as possible without detrimentally affecting the quality of the powder. A falling film evaporator has low residence times (typically 3–6 min) and operates under vacuum so that boiling temperatures range from 40 to 70°C. About 80% of the water can be removed from milk, concentrating it from 9–14% total solids up to 44–50% total solids. Evaporation causes a number of changes in the milk system (Singh and Newstead, 1992). The pH of the milk decreases during concentration from an average initial value of 6.7 to approximately 6.3 at 45% total solids. This is partly due to changes in salt equilibria as more calcium phosphate is transferred from the soluble state to the colloidal state, with a concomitant release of hydrogen ions. There is no significant denaturation of β-LG or Ig G during the evaporation of skim milk to 45% total solids, whereas some denaturation of α-LA may occur (Oldfield et al., 2005). This is considered to be partly due to the increased stability of whey proteins at high total solids concentrations, because of the stabilizing effect of lactose on the protein structure. However, there is an increase in the amounts of β-LG and α-LA associated with the micelles after evaporation, which can be attributed mainly to the decrease in pH during evaporation (Oldfield et al., 2005).

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Dairy-derived ingredients

Casein micelles may increase in size because of the increase in CCP or increased association of the whey proteins with the casein micelles. Unfortunately, no reliable data on the size distribution of casein micelles in highly concentrated systems are available, mainly because of the nonavailability of measurement techniques. In addition, concentrated milk is very difficult to handle because of its high viscosity and its tendency towards age gelation. Under normal conditions with the evaporation temperatures below 70°C, the alterations to the proteins are likely to be minor compared with those incurred during preheating (Singh and Creamer, 1991a). 25.3.3 Concentration of skim milk by ultra- and diafiltration Ultrafiltration (UF) is a pressure-driven filtration process in which porous membranes are used to separate the components of a solid–liquid mixture on the basis of size and shape. UF membranes are available in many forms but tubular membranes are most popular in modern dairy UF plants. These membranes are available with wide ranges of porosity and molecular mass: UF of skim milk is commonly carried out using membranes with a molecular weight cut-off of 10 000 Da. By this process, a concentrate of high molecular weight (retentate) and a permeate fraction of low molecular weight are obtained. UF processing of skim milk allows water, lactose, nonprotein nitrogen compounds and soluble salts to pass through the membrane while retaining casein micelles and whey proteins. Some calcium, magnesium, phosphate and citrate are associated with the casein micelles in milk, and hence are retained in the concentrate. The temperature of UF has an impact on the composition of the concentrate, with higher temperatures resulting in higher permeation rates and lower retention of lower molecular weight components. Diafiltration (DF) is employed to produce milk powders with high protein contents, typically over 80%. Purified water is commonly used. The number of DF stages and the volume of water used are dependent on the design and operation of the UF plant and the final product specification. Relatively little work on the behaviour of the casein micelles during UF/ DF has been reported. Srilaorkul et al. (1991), using electron microscopy, showed that the size of the highest proportion of casein micelles (80–100 nm) in milk was reduced (to 60–80 nm) at a volume concentration factor of about 5. These changes in micelle size were considered to be due mainly to the changes in the levels of milk salts, particularly calcium and phosphate. In contrast, McKenna (2000) found that there was little change in particle size in the early stages of UF, but that the average micelle size increased significantly at the end of the UF process, and particularly during DF. McKenna (2000) observed a progressive swelling of the casein micelles and the formation of non-micellar material. Figure 25.3 shows a freeze fracture transmission electron micrograph of skim milk; roughly spherical particles, i.e. casein micelles, of different sizes

Protein interactions and functionality of milk protein products (a)

(c)

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(b)

(d)

Fig. 25.3 Freeze fracture transmission electron microscopy of (a) skim milk, (b) ultrafiltrated skim milk (mid-way), (c) ultrafiltrated skim milk and (d) diafiltrated skim milk (From McKenna, 2000).

are clearly observed (Fig. 25.3a). Mid-way through and after UF, there appears to be no considerable change in casein micelle size, but more nonmicellar material can be seen in the micrographs (arrows) (Fig. 25.3b,c). After DF, a significant change in the structure of the casein micelles appears to take place, with an increase in non-micellar material, and the casein micelle appears to be swollen and less dense (Fig. 25.3d). The non-micellar material appears to link these micelles together. The dissolution of CCP results in loosening of the casein micelle structure and is thus responsible for the swelling of the micelles. 25.3.4 Spray drying Spray drying removes the remaining water in the concentrate to produce a powder with 3–5% moisture. Milk concentrate is atomized into a fine spray that gives a large surface area over which drying can take place. In the drying chamber, the droplets are intimately mixed with the incoming hot air (150–250°C). Initially, milk droplets dry rapidly at their wet bulb temperature (45–60°C); however, as the droplets progress down the drying chamber, they become drier and the particle temperature eventually approaches the air temperature at the drier outlet (70–90°C).

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There are a number of different drier configurations, with or without internal and external fluidized beds. Two-stage and multi-stage driers are more energy efficient and less damaging to the properties of milk powder than single-stage driers. The drying conditions influence powder properties: moisture content, solubility, bulk density and powder flowability (Pisecky, 1997). The outlet temperature has a marked effect on powder solubility. Increasing the outlet temperature results in poor powder solubility. However, little is known about the changes to proteins that occur during spray drying. The native properties of the milk components are essentially unmodified by moderate drying conditions. The normal size distribution of the casein micelles and their heat stabilities and renneting characteristics are substantially recovered on the reconstitution of spray-dried milk. Under typical drying conditions, whey proteins are not denatured to any great extent. Oldfield et al. (2005) showed that the denaturation of whey proteins during spray drying was minimal with no apparent loss of Ig G and only a small loss of BSA (3–7%). Varying the inlet/outlet drier air temperature (200/100°C–160/89°C) appears to have no significant effect on whey protein denaturation. However, high outlet temperature conditions (>80°C) can cause lactosylation of milk proteins (part of the Maillard reaction between proteins and lactose), decreasing their nutritional value (Guyomarc’h et al., 2000). Spray drying would be expected to cause some changes in the salt equilibria, possibly an increase in CCP and a decrease in pH. It has been shown that the concentrations of soluble calcium and soluble phosphate in reconstituted skim milk are about 20% lower than those in the original milk (Le Graet and Brule, 1982). Roller driers can also be used to produce milk powders, but they are limited to producing whole milk powders for confectionary, especially chocolate, manufacture (Augustin, 2001). Roller-dried whole milk powders have a high level of free fat and low solubility, which make them ideal for use in chocolate.

25.4

Protein interactions during the manufacture of whey protein and casein products

25.4.1 Whey protein products Sweet or acid whey, derived from cheese or casein production, is used for the manufacture of WPCs and WPIs. WPIs can also be manufactured directly from skim milk using microfiltration technology. There is a range of whey pretreatment processes, such as clarification, preheating and pasteurization, that may be carried out to improve flux and to reduce fouling during UF, and to manipulate the chemical composition of the final product. The purpose of centrifugal clarification is to remove

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particulate matter such as fat, casein fines and microorganisms. Preheating of whey to at least the temperature of UF is carried out to improve the efficiency of the UF process. These processes are unlikely to have an effect on the native structures of the whey proteins. The clarified whey, particularly in the case of cheese whey, is commonly pasteurized at 72°C for 15 s, which is likely to cause denaturation of minor, heat-sensitive whey proteins, such as Igs and BSA. In comparison, acid whey is generally not pasteurized because such a heat treatment at the natural pH of acid whey (pH ~4.6) can result in protein aggregation (Hobman, 1992). UF and DF processes of whey are commonly carried out at ~50°C. This temperature is generally considered to be optimum; acceptable fluxes are achieved and thermal denaturation of the whey proteins is avoided. The evaporation of whey retentate is carried out using high vacuum, low boiling temperature (e.g. 50°C) falling film evaporators, which enable effective concentration of the retentate up to 40% total solids (Nielsen, 1988), while avoiding thermal denaturation of the whey proteins. Drying of the retentate is usually performed using a spray drier fitted with nozzle atomization. Typically, the inlet and outlet air temperatures used are 160–180°C and less than 80°C, respectively (Hobman, 1992). As in the case of milk powder drying, the temperature of the drying droplets does not exceed 70°C, ensuring no damage to the proteins. de la Fuente et al. (2002) examined the structural changes in proteins during the major steps of WPC manufacture, using size exclusion chromatography combined with multi-angle laser light scattering (SEC–MALLS). Samples of acid whey and cheese whey were obtained from commercial plants, throughout the WPC process as follows: whey after clarification; retentate mid-way through UF/DF; retentate after DF; concentrate after evaporation; and WPC after spray drying. There were some differences in the protein compositions between the two types of whey, with the acid casein whey containing lower proportions of ‘soluble’ aggregates than the cheese whey. UF/DF processes caused a removal of non-protein, low molecular weight components, but had no significant effect on protein composition and aggregation. Evaporation and spray drying of the retentates caused no significant changes in protein denaturation and aggregation. The presence of small amounts of protein aggregates in WPC products results from heat treatments applied to milk or whey (mainly pasteurization) prior to UF/DF and drying. Therefore, it appears that variability in protein denaturation between WPCs is likely to arise from heat treatments used in casein and cheese manufacture rather than from the WPC manufacturing process. 25.4.2 Casein products The manufacture of acid casein involves the isoelectric precipitation of casein from skim milk, either by direct addition of mineral acid or by

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acidification by bacterial cultures that ferment lactose to lactic acid. During the acidification of milk, many of the physico-chemical properties of casein micelles undergo considerable changes. As the pH of skim milk is reduced, CCP is dissolved and the caseins are dissociated into the milk serum phase (Roefs et al., 1985; Dalgleish and Law, 1988). The extent of dissociation of the caseins is dependent on the temperature of acidification; at 30°C, a decrease in pH causes virtually no dissociation; at 4°C, about 40% of the caseins are dissociated in the serum at pH ~5.5 (Dalgleish and Law, 1988). Apparently little change in the average hydrodynamic diameter of the casein micelles occurs during acidification of the milk to pH ~5.0 (Roefs et al., 1985). The lack of change in the size of the micelles on reducing the pH of milk to 5.5 may be due to concomitant swelling of the particles as CCP is solubilized. Aggregation of casein occurs as the isoelectric point (pH 4.6) is approached. The pH and the temperature of the curd and whey at separation determine the properties of the curd (Jablonka and Munro, 1985). These effects appear to be related to the level of residual calcium in the curd. Below about pH 4.4, almost all of the calcium is lost from the micelle at any temperature but above pH 4.4, the loss of calcium is temperature sensitive with more calcium being retained at higher temperatures. The curd strength follows the same trend, with high calcium being comparable with high cohesiveness of the curd. There is an ideal cohesiveness and an ideal calcium concentration, which give springy non-sticky curd that holds together. If the curd is produced at too acid a pH, it lacks cohesiveness but this can be improved to some degree by using a higher cooking temperature. If not enough acid is added, the curd will be too cohesive (tough and unwashable and may be sticky) but this can be improved to some degree by using a lower cooking temperature. In the manufacture of caseinate, fresh acid casein curd or dry acid casein is solubilized in a dilute solution of alkali and then spray dried, as described earlier. Manufacturing processes for sodium caseinate can vary from producer to producer. Differences in the chemical composition between batches of sodium caseinate from the same and different manufacturers have been found. Lynch et al. (1997) reported some differences in the profiles obtained by SEC on different batches of sodium caseinate. Lucey et al. (2000) examined a range of commercial and laboratory-made sodium caseinate samples using a MALLS system. Sodium caseinate solutions, analyzed using a MALLS system alone, gave weight-average molar mass (Mw) values in the range 1200–4700 kDa and the z-average root-meansquare radius (Rg) values ranged from ~50 to 120 nm. When these solutions were ultracentrifuged at 90 000 g for 1 h, and the subnatants analyzed by SEC–MALLS, the Mw values were found to be in the range ~30–575 kDa and the Rg values ranged from ~22 to 49 nm. Laboratory-made (never dried) sodium caseinate samples had the smallest Mw values, suggesting some aggregation of casein molecules during the drying process.

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In the manufacture of rennet casein, the coagulation of milk occurs in phases: a primary enzymatic phase, a secondary non-enzymatic phase and a less clearly defined tertiary phase (Dalgleish, 1992). During the primary phase, rennet causes specific hydrolysis of κ-casein in the region of the phenylalanine105–methionine106 bond, Phe105 supplying the carboxyl group and Met106 supplying the amino group, resulting in the formation of two peptides of contrasting physical and chemical properties. The glycomacropeptide (GMP) moiety, which comprises amino acid residues 106–169, is hydrophilic and diffuses away from the casein micelle after splitting from κ-casein and into the serum. The second peptide, para-κ-casein, which consists of amino acid residues 1–105, is strongly hydrophobic and remains attached to the micelles. κ-casein hydrolysis during the primary phase alters the properties of the casein micelles, rendering them susceptible to aggregation and this marks the onset of the secondary phase. Loss of GMP during the primary phase of renneting results in loss of about half the negative charge as well as surface steric repulsion of κ-casein (Dalgleish, 1984). Consequently, the micelle surface becomes more hydrophobic because of the accumulation of paraκ-casein, and micelle aggregation becomes possible. There is a critical value of κ-casein hydrolysis (86–88%) below which the micelles cannot aggregate. The action of rennet can be seen as providing ‘hot spots’ (areas on the casein micelle surfaces from which the protective GMP moiety has been ‘shaved off’) via which the micelles can aggregate, these reactive areas being produced by the removal of κ-casein from a sufficiently large area (Dalgleish, 1992). As the last of the stabilizing surface is removed (i.e. during the destruction of the final 10% of the κ-casein), the concentration of micelles capable of aggregation and the rate at which they aggregate increase rapidly. The process of gel assembly during the secondary phase of rennet coagulation appears to involve the formation of small aggregates in which the micelles tend to be joined in chains that link together randomly to form a network (Green et al., 1978). Initially, linkage of aggregated micelles is through bridges, which slowly contract with time, forcing the micelles into contact and eventually causing them to partly fuse. This process probably progressively strengthens the links between micelles, giving an increase in curd firmness after coagulation. The tertiary stage involves the rearrangement of the network and processes such as syneresis and the non-specific proteolysis of the caseins in the rennet gel (Dalgleish, 1992).

25.5

Functional properties and applications of milk protein products

25.5.1 Skim milk powders SMPs are used as ingredients in a range of other dairy foods, in particular in recombined milk products. SMP is generally mixed with water to the

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required concentration and other ingredients are added, followed by further processing and packaging. Various types of recombined milk products, including recombined pasteurized milk, recombined evaporated milk (REM), recombined sweetened condensed milk, yoghurt, cheese and ice cream, are manufactured using SMP. In fact, recombined analogues of all dairy products made from fresh milk can be produced. Among others, the functional properties of SMP in these products include solubility, heat stability, rennet coagulation, acid gelation and thickening. These properties are determined by the physical and chemical properties of the primary constituents, namely proteins and lactose, both individually and in combination, which in turn are affected by the extent of protein interactions during powder manufacture. As the heat stability of SMP is one of the most important functional properties in most applications, the following section discusses some key aspects of this property. Heat stability The heat stability of milk refers to the ability of milk to withstand high processing temperatures without visible coagulation or gelation or a large increase in viscosity. The heat stability characteristics of reconstituted SMP are important when milk powders are used in the manufacture of REM or as ingredients in hot beverages, such as custards and soups. However, most of the commercial heat stability problems are concerned with recombined concentrated products (e.g. REM). The total solids content of REM ranges from 26 to 31% and these milks are typically sterilized at 120°C for 12–23 min. Milk powders reconstituted to normal milk solids (9%) and subjected to UHT-type heat treatments or even sterilization heat treatments (120°C) are generally stable. The heat stability aspects of skim milk have been studied by several workers and have been reviewed extensively (Fox, 1982; Singh and Creamer, 1992; O’Connell and Fox, 2003). A number of factors influence the heat stability of milk (both normal and concentrated). The factors affecting the heat stability of fresh milk and reconstituted and recombined milks are very similar and hence the vast amount of information available on fresh milk can be applied directly to recombined milks. The heat stability of milk is affected by the mineral balance, the lactose, urea and protein contents and the milk processing parameters, such as preheating and homogenization. Fat per se has no effect on the heat stability because skim milk is as stable to heat as the whole milk from which it is prepared. However, when fat-containing milk is subsequently homogenized, a clear decrease in heat stability is observed. Some of the important factors, particularly those relevant in the manufacture of recombined milks, are described next. The heat coagulation time (HCT) of fresh skim milk at 140°C is ~14–19 min and is markedly affected by pH. A plot of HCT versus pH

Protein interactions and functionality of milk protein products

661

40

HCT at 140 °C

35 30 25 20 15 10 5 0 6.2

6.4

6.6

6.8

7.0

7.2

7.4

pH

Fig. 25.4 Heat coagulation time (HCT) versus pH profile for normal skim milk (䉱) or concentrated milk (20% total solids) (•) heated at 140°C.

typically shows a maximum at around pH 6.6–6.7 and a minimum in the pH region 6.8–6.9 (Fig. 25.4). The heat stability of fresh or reconstituted concentrated milk is much lower than that of unconcentrated milk but the effect of pH is similar, with the maximum heat stability usually occurring in the pH range 6.4–6.6. However, concentrated milk is unstable at all pH values above 6.8 (Fig. 25.4). It is widely recognized that preheating improves the heat stability of milk, especially when the product is subsequently concentrated. Preheat treatments of 80–90°C with holding times of 10–20 min are commonly used. However, it has been shown that preheating temperatures in excess of 100°C with shorter holding times provide better stability in the concentrated product. Studies in New Zealand on the effects of preheat treatment on the heat stability of REM have shown that heat treatments of 120°C for 120 s, prior to evaporation and drying of the SMP, result in a heat-stable REM (Newstead et al., 1975). Longer periods of preheating at 120°C can further improve the heat stability during the early and late parts of the season, but decrease the heat stability during the mid-season (Singh and Tokley, 1990). Soluble salts, particularly calcium and phosphate, play an important role in the heat stability of milk. Generally, the addition of calcium salts to milk decreases the heat stability, whereas a reduction in calcium has a stabilizing effect. Concentrated milks with lower levels of soluble salts (e.g. concentrates prepared by UF) are markedly more stable that those prepared by standard evaporation. The addition of sodium or potassium salts of orthophosphoric and citric acids is permitted in many countries and increases the heat stability of concentrated milk. The addition of commercial phosphates

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Dairy-derived ingredients

(mixtures of NaH2PO4, Na2HPO4 and Na3PO4) improves the heat stability of concentrated milk and is a common practice in the recombined milk industry (Augustin, 2000). Urea concentration is a major factor affecting the heat stability of skim milk. Addition of urea improves the heat stability of unconcentrated milks but has no effect on the heat stability of concentrated milks. Addition of formaldehyde to normal milk and concentrated milk increases the heat stability. The heat stability of recombined milk may be improved by incorporating buttermilk powder (BMP) and lecithin during homogenization (Singh and Tokley, 1990; McCrae and Muir, 1992). BMP improved the heat stability of REM during most of the dairying season (Singh and Tokley, 1990). Lecithin can be added at different stages of the manufacturing process: (i) before preheating, (ii) after preheating but before homogenization, and (iii) immediately after homogenization. Polyphenol-rich extracts of tea, coffee, cocoa, wine, pine bark and oak leaves and several pure phenolic compounds increase the heat stability of both unconcentrated milk and concentrated milk (O’Connell et al., 1998). However, the effects of these compounds on the sensory properties of the milks are not known. Mechanisms of heat coagulation The coagulation of milk on extended heating at high temperatures (120– 140°C) is a consequence of the loss of casein micelle stability, as a result of numerous physical and chemical changes in its components. Casein micelles are stabilized primarily by the surface layer of κ-casein, which provides steric stabilization and contributes the overall negative charge. Inside the casein micelle, the individual casein molecules are associated by hydrophobic and electrostatic bonds in which CCP also plays an important role (Holt, 1992). Heat treatment markedly changes the environment around the casein micelles (i.e. serum phase) (e.g. change in pH and soluble minerals, in particular calcium ions, breakdown of lactose and urea) as well as the casein micelles themselves (association of whey proteins, changes in CCP, dephosphorylation, casein dissociation). It is not known exactly which particular changes are directly responsible for coagulation, predispose the milk to coagulation or are a consequence of the coagulation process. For more detailed discussion on this topic, refer to Singh and Creamer (1992), O’Connell and Fox (2003) and Singh (2004). The pH dependence of heat stability has received considerable attention, and it is now believed that the occurrence of a maximum (pH around 6.7) in the HCT–pH profile of normal milk is essentially caused by whey protein interactions, through the formation of a complex between κ-casein and β-LG on the surface of the casein micelles, thus altering the steric and electrostatic interactions (Singh and Fox, 1987; van Boekel et al., 1989; Singh and Creamer, 1992; Singh, 2004). Although the exact mechanism of the coagulation of whey-protein-coated micelles is not clear, it is likely that

Protein interactions and functionality of milk protein products

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further heating causes lowering of the pH, dephosphorylation, covalent bond formation and other reactions. As a result, the altered micelles are able to make more frequent contact and probably form covalent cross-links, thus promoting micelle aggregation. At pH below 6.7, the protective effect of the complex is insufficient to overcome the effects of increasing acidity and high calcium ion concentrations. At pH above 6.9, the stability decreases due to dissociation of micellar κ-casein, thus reducing the stabilizing effect of κ-casein (Singh and Fox, 1987; van Boekel et al., 1989). The κ-casein-depleted micelles are sensitive to calcium ion concentrations. Therefore, the minimum in the HCT–pH profile is a result of coagulation of κ-casein-depleted micelles; coagulation is essentially salt induced, caused by calcium ions. At higher pH, although the dissociation of micellar κ-casein increases, the HCT increases because of the increase in protein charge and the low calcium ion activity. When concentrated milk is heated at its normal pH, there is initial association of whey proteins with the micellar κ-casein. This is followed by dissociation of κ-casein-rich protein from the micelles, which creates sites on the micelles that are favourable for micelle–micelle interactions. Thus, in concentrated milk, coagulation is due to the sensitivity of κ-caseindepleted micelles to calcium ions, a situation that is somewhat similar to that existing for the coagulation of normal milk within the region of minimum stability. At high pH, the micelles are highly dissociated and coagulation is more like a gelation, with the dissociated material acting as a bridging agent (Nieuwenhuijse et al., 1991; Singh and Creamer, 1991b; Singh et al., 1995). Production of heat-stable milk powders – practical considerations The problems relating to the production of heat-stable milk powders (i.e. milk powders that are stable to heat sterilization after reconstitution to form REM) largely relate to seasonal variations and batch-to-batch variations in heat stability. Practical solutions to the heat stability problems include the following: Manipulation of preheat treatments – Production of heat-stable SMP is primarily controlled by providing a sufficient preheat treatment, before evaporation, to denature the whey proteins. High heat treatments are typically of the order of 115–125°C for 1–3 min. However, high heat treatments are not a guarantee of producing heat-stable SMP. In countries with seasonal variations in milk composition (e.g. New Zealand, Australia, Ireland), milk from the middle of the season is used, as milk at the beginning and the end of the season (late lactation milk) tends to have lower heat stability (Kelly et al., 1982). It is possible to make heatstable SMP from late lactation milk by careful screening of powders and control of processing conditions (Singh and Newstead, 1992). Addition of different levels of phosphate salts – Sodium and potassium salts of orthophosphate and citrate are used to improve the heat stability of

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REM. Care should be taken as over-use of salts can cause the heat stability to become worse. Thus, heat stability tests are used to select the right combination and level of salts. Food regulations limit the amount of stabilizing salt that can be added – besides the limit of not producing a salty product. In order for stabilizing salts to work, the overall heat stability maximum needs to be increased and, more importantly, the pH of the REM should be positioned on or close to this maximum. For example, di-sodium hydrogen orthophosphate (DSP) and mono-sodium dihydrogen orthophosphate (MSP) are commonly used to improve heat stability. Addition of either salt shifts the heat stability curve to a more acidic pH and increases the heat stability maximum. The sodium salts also change the pH: DSP increases the natural pH, whereas MSP reduces the natural pH. Thus, salt selection depends on which side of the maximum the pH of REM is. Often, small pH shifts are required to position the pH of REM at the maximum, so that combinations of MSP and DSP are used. It is also possible to incorporate appropriate amounts of stabilizing phosphate salts into the milk prior to drying. Addition of buttermilk and phospholipids at appropriate levels – BMP can be used to replace up to 10% of the SMP in REM. Phospholipids in the buttermilk improve the heat stability, contribute to the creamy flavour, and help to emulsify the fat (Singh and Tokley, 1990). Alternatively, buttermilk can be incorporated into milk powders before or after evaporation, to improve their heat stability.

25.5.2 Milk protein powders The milk protein products, such as WPC, WPI, MPC, are added to a formulation for a particular property, either nutritional or functional. The functional properties of proteins in a vast range of food products include water absorption, solubility, viscosity, gelation, emulsification, foaming and film formation. Some important functional properties of milk protein products are shown in Table 25.4. The functional properties of milk protein products may be considered to be the consequence of the modifications of molecular structures and their interactions. Therefore, the modification of native protein structures and their consequent interactions with other food components, such as lipids, non-dairy proteins and carbohydrates, can exert a negative or positive effect on the functional properties of milk protein products. The functional properties of milk proteins vary with pH, temperature, ionic strength, and concentration of calcium and other polyvalent ions, sugars and hydrocolloids, as well as with processing treatments. Some of the key functional properties, such as solubility, emulsification and gelation, are discussed in this section. For more extensive information on the functional properties of milk proteins, the reader is referred to de Wit (1989), Kinsella and Whitehead (1989), Mulvihill (1992) and Mulvihill and Ennis (2003).

Protein interactions and functionality of milk protein products Table 25.4

665

Key functional properties of different milk protein products Whey protein products

Caseinates

Solubility Emulsification Foaming Water binding Viscosity Gelation Heat stability Acid stability Freeze/thaw stability

Milk protein concentrates

Sodium

Calcium

WPC

WPI

MPC

xxx xxx xxx xxx xxx – xxx x xxx

x x x x x – x x x

xxx xx xx x x xxx x xxx x

xxx xx xx x x x x xxx x

xx x x x x – x x x

x: poor, xx: good, xxx: excellent.

Solubility Solubility is generally defined as the amount of protein in a sample that goes into solution or into colloidal dispersion under specified conditions and is not sedimented by low centrifugal forces. The most common methods for determining solubility involve the preparation of a protein solution of known concentration (0.5–5.0%), centrifugation at forces ranging from 500 to 65 000 g for 5–40 min and then protein analysis of the supernatant. The solubility of milk protein products depends on the nature of the proteins in the product, the processing treatments used, the physical form of the powder, pH, and the temperature and ionic concentration of the solution. In general, high temperature treatments during the manufacturing process result in low solubility of the final product. Salts may enhance the solubility of protein up to a point and then decrease it. The pH affects the charge and electrostatic interactions between protein molecules. Above and below the isoelectric point, proteins have a net negative or positive charge and enhanced solubility. At the isoelectric point, attractive forces predominate, causing protein molecules to associate, resulting in a loss of solubility. SMPs generally have excellent solubility in water under a wide range of conditions, except at close to the isoelectric point of casein. MPCs with up to 70% protein have good solubility at pH 7.0, but MPC powders with very high protein content (e.g. MPC85) are generally known to have poor solubility upon reconstitution in water at 20°C. The solubility of these powders improves at higher reconstitution temperatures, but decreases with storage time at elevated temperatures (McKenna, 2000). The major factor affecting the solubility behaviour of MPC85 appears to be related to the

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rate of water transfer into the powder particle rather than to the thermal processes during manufacture (Schuck et al., 1994). Various methods for manufacturing MPC powders with improved solubility in cold water have been proposed, involving the addition of a monovalent salt to the ultrafiltered retentate prior to drying (Carr, 2002) or partial replacement (~30%) of the calcium content of ultrafiltered retentate by sodium ions (Bhaskar et al., 2001). The insoluble material found in reconstituted MPC powders has been characterized by McKenna (2000) and Havea (2006). Electron microscopy showed that the insoluble material in MPC85 consists of large particles (up to 100 µm) formed by the fusion of casein micelles, involving some kind of protein–protein interactions (McKenna, 2000). These fused casein micelles appear to form a skin-like structure on the outside of the powder particle, inhibiting the movement of water into the particle. Upon reconstitution in water, large parts of these particles remain intact. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the insoluble material consisted predominantly of αs- and β-caseins, and that this material was held together by weak non-covalent interactions that were easily disrupted under the PAGE conditions (Havea, 2006). Some disulphide-linked protein aggregates consisting of κ-casein and β-LG were present in the MPC powders, but these aggregates were not considered to play an important role in the formation of insoluble material. Further work is required to understand the nature of the protein–protein interactions involved in the formation of insoluble material, and to develop methods that can be used to minimize protein interactions during the manufacture of MPC. Rennet and acid caseins are insoluble in water but caseinates (sodium, potassium and ammonium) have excellent solubility. However, caseinates are insoluble in the isoelectric (pH 4.0–5.0) region, but they are soluble at pH below 3.5. Calcium caseinate forms a coarse colloidal dispersion under these conditions (see Mulvihill and Ennis, 2003). Whey proteins show excellent solubility over the entire pH range, but heat-denatured whey proteins are insoluble in the pH range 4–5. Processing treatments used in the manufacture of WPCs and WPIs may sometimes cause small amounts of denaturation, which tends to reduce their solubility (de Wit and Klarenbeek, 1984; de Wit et al., 1996). The solubility of whey proteins is generally not altered by UF or spray drying, although pasteurization may cause up to 20% denaturation with a consequent loss of solubility. Emulsification As milk proteins, both caseinates and whey proteins, are surface active, they are adsorbed rapidly to the oil–water interface, forming stable oil-inwater emulsions. Sodium caseinate and WPC/WPI show excellent emulsifying abilities. In emulsions formed with sodium caseinate or WPC, the

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surface protein concentration increases with an increase in protein concentration until it reaches a plateau value of about 2.0–3.0 mg/m2 (Singh, 2005; Singh and Ye, 2009). The emulsifying ability of ‘aggregated’ milk protein products, such as MPC and calcium caseinate, is much lower than that of WPC or sodium caseinate, i.e. much higher concentrations of protein are required to make stable emulsions and larger droplets are formed under similar homogenization conditions (Euston and Hirst, 1999). The surface protein concentration of emulsions formed with MPC is in the range 5–20 mg/m2 depending on the protein concentration used in making the emulsions. When the ratio of protein to oil is low in these emulsions, protein aggregates are shared by adjacent droplets, resulting in bridging flocculation and consequently a marked increase in droplet size. In sodium-caseinate-based emulsions, there is competition between individual proteins for adsorption on to the oil–water interface during emulsification. β-Casein appears to adsorb in preference to αs1-casein at low protein concentrations (